Recombinant adeno-associated virus capsids resistant to pre-existing human neutralizing antibodies

ABSTRACT

The present invention relates to variant AAV capsid polypeptides, wherein the variant capsid polypeptides exhibit an enhanced neutralization profile, increased transduction and/or tropism in human liver tissue or hepatocyte cells (i.e., human hepatocyte cells), or both, as compared non-variant parent capsid polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/435,212, filed Feb. 16, 2017, which claims the benefit of U.S.Provisional Application No. 62/296,046, filed on Feb. 16, 2016, all ofwhich is expressly incorporated herein by reference in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under contractsHL092096, AI116698, OD010580 and HL119059 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to variant AAV capsid polypeptides,wherein the variant capsid polypeptides exhibit an enhancedneutralization profile, exhibit increased transduction or tropism inhuman liver tissue or hepatocyte cells (i.e., human hepatocyte cells),or both as compared to non-variant parent capsid polypeptides.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM,LISTING APPENDIX SUBMITTED ON A COMPACT DISK

This invention incorporated by reference the Sequence Listing text copysubmitted herewith, which was created on Apr. 15, 2017, entitled068597_5031_US_ST25.txt which is 61 kilobytes in size.

BACKGROUND OF THE INVENTION

Genetic disorders caused by absence of or a defect in a desirable gene(loss of function) or expression of an undesirable or defective gene(gain of function) lead to a variety of diseases. At present,adeno-associated virus (AAV) vectors are recognized as the gene transfervectors of choice for therapeutic applications since they have the bestsafety and efficacy profile for the delivery of genes in vivo. Of theAAV serotypes isolated so far, AAV2, AAV5, AAV6 and AAV8 have been usedto target the liver of humans affected by severe hemophilia B. In thecase of AAV8, long-term expression of the therapeutic transgene wasdocumented. Recent data from humans showed that targeting the liver withan AAV vector can achieve long-term expression of the FIX transgene attherapeutic levels. Additionally, several Phase 1 and Phase 2 clinicaltrials using various AAV serotypes have been reported for the treatmentof alpha-1 antitrypsin deficiency (M. L. Brantly, J. D. Chulay, L. Wang,C. Mueller, M. Humphries, L. T. Spencer, F. Rouhani, T. J. Conlon, R.Calcedo, M. R. Betts, C. Spencer, B. J. Byrne, J. M. Wilson, T. R.Flotte, Sustained transgene expression despite T lymphocyte responses ina clinical trial of rAAV1-AAT gene therapy. Proceedings of the NationalAcademy of Sciences of the United States of America 106, 16363-16368(2009); T. R. Flotte, B. C. Trapnell, M. Humphries, B. Carey, R.Calcedo, F. Rouhani, M. Campbell-Thompson, A. T. Yachnis, R. A.Sandhaus, N. G. McElvaney, C. Mueller, L. M. Messina, J. M. Wilson, M.Brantly, D. R. Knop, G. J. Ye, J. D. Chulay, Phase 2 clinical trial of arecombinant adeno-associated viral vector expressing alphal-antitrypsin:interim results. Human gene therapy 22, 1239-1247 (2011); C. Mueller, J.D. Chulay, B. C. Trapnell, M. Humphries, B. Carey, R. A. Sandhaus, N. G.McElvaney, L. Messina, Q. Tang, F. N. Rouhani, M. Campbell-Thompson, A.D. Fu, A. Yachnis, D. R. Knop, G. J. Ye, M. Brantly, R. Calcedo, S.Somanathan, L. P. Richman, R. H. Vonderheide, M. A. Hulme, T. M. Brusko,J. M. Wilson, T. R. Flotte, Human Treg responses allow sustainedrecombinant adeno-associated virus-mediated transgene expression. TheJournal of clinical investigation 123, 5310-5318 (2013)). Additionally,numerous world-wide trials are underway, including for example, trialES-0020 with AAV5 for acute intermittent porphyria (AIP) (Phase I);IE-0001 with AAV1 for hAAT (alpha-1 antitrypsin) (Phase II); NL-0037with AAV5 for hemophilia B (Phase I); UK-0137 with AAV2 for hemophilia B(Phase I); US-0864 with AAV2 for hemophilia B (Phase I); US-1441 withAAV8 for hemophilia B (Phase I/II); US-1355 with AAV8 for hemophilia B(Phase I/II); US-1144 with AAV8 for hypercholesterolemia (Phase I);US-1398 with AAVrh10 for hemophilia B (Phase I/II); US-1446 withAAV2/AAV6 for hemophilia B (Phase I); and US-1520 with AAV8 forLate-Onset Ornithine Transcarbamylase (OTC) deficiency (Phase I/II).See, the World Wide Web at abedia.com/wiley/index.html.

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icosahedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb). AAV is assigned to the genus,Dependovirus, because the virus was discovered as a contaminant inpurified adenovirus stocks (D. M Knipe, P. M Howley, Field's Virology,Lippincott Williams & Wilkins, Philadelphia, ed. Sixth, 2013). In itswild-type state, AAV depends on a helper virus—typically adenovirus—toprovide necessary protein factors for replication, as AAV is naturallyreplication-defective. The 4.7-kb genome of AAV is flanked by twoinverted terminal repeats (ITRs) that fold into a hairpin shapeimportant for replication. Being naturally replication-defective andcapable of transducing nearly every cell type in the human body, AAVrepresents an ideal vector for therapeutic use in gene therapy orvaccine delivery. In its wild-type state, AAV's life cycle includes alatent phase during which AAV genomes, after infection, are sitespecifically integrated into host chromosomes and an infectious phaseduring which, following either adenovirus or herpes simplex virusinfection, the integrated genomes are subsequently rescued, replicated,and packaged into infectious viruses. When vectorized, the viral Rep andCap genes of AAV are removed and provided in trans during virusproduction, making the ITRs the only viral DNA that remains (A.Vasileva, R. Jessberger, Nature reviews. Microbiology, 3, 837-847(2005)). Rep and Cap are then replaced with an array of possibletransfer vector configurations to perform gene addition or genetargeting. These vectorized recombinant AAVs (rAAV) transduce bothdividing and non-dividing cells, and show robust stable expression inquiescent tissues. The number of rAAV gene therapy clinical trials thathave been completed or are ongoing to treat various inherited oracquired diseases is increasing dramatically as rAAV-based therapiesincrease in popularity. Similarly, in the clinical vaccine space, therehave been numerous recent preclinical studies and one ongoing clinicaltrial using rAAV as a vector to deliver antibody expression cassettes inpassive vaccine approaches for human/simian immunodeficiency virus(HIV/SIV), influenza virus, henipavirus, and human papilloma virus(HPV). (See, P. R. Johnson, B. C. Schnepp, J. Zhang, M. J. Connell, S.M. Greene, E. Yuste, R. C. Desrosiers, K. R. Clark, Nature medicine 15,901-906 (2009); A. B. Balazs, J. Chen, C. M. Hong, D. S. Rao, L. Yang,D. Baltimore, Nature 481, 81-84 (2012); A. B. Balazs, Y. Ouyang, C. M.Hong, J. Chen, S. M. Nguyen, D. S. Rao, D. S. An, D. Baltimore, Naturemedicine 20, 296-300 (2014); A. B. Balazs, J. D. Bloom, C. M. Hong, D.S. Rao, D. Baltimore, Nature biotechnology 31, 647-652 (2013); M. P.Limberis, V. S. Adam, G. Wong, J. Gren, D. Kobasa, T. M. Ross, G. P.Kobinger, A. Tretiakova, J. M., Science translational medicine 5,187ra172 (2013); M. P. Limberis, T. Racine, D. Kobasa, Y. Li, G. F. Gao,G. Kobinger, J. M. Wilson, Vectored expression of the broadlyneutralizing antibody F16 in mouse airway provides partial protectionagainst a new avian influenza A virus, H7N9. Clinical and vaccineimmunology: CVI 20, 1836-1837 (2013); J. Lin, R. Calcedo, L. H.Vandenberghe, P. Bell, S. Somanathan, J. M. Wilson, Journal of virology83, 12738-12750 (2009); I. Sipo, M. Knauf, H. Fechner, W. Poller, O.Planz, R. Kurth, S. Norley, Vaccine 29, 1690-1699 (2011); A. Ploquin, J.Szecsi, C. Mathieu, V. Guillaume, V. Barateau, K. C. Ong, K. T. Wong, F.L. Cosset, B. Horvat, A. Salvetti, The Journal of infectious diseases207, 469-478 (2013); D. Kuck, T. Lau, B. Leuchs, A. Kern, M. Muller, L.Gissmann, J. A. Kleinschmidt, Journal of virology 80, 2621-2630 (2006);K. Nieto, A. Kern, B. Leuchs, L. Gissmann, M. Muller, J. A.Kleinschmidt, Antiviral therapy 14, 1125-1137 (2009); K. Nieto, C.Stahl-Hennig, B. Leuchs, M. Muller, L. Gissmann, J. A. Kleinschmidt,Human gene therapy 23, 733-741 (2012); and L. Zhou, T. Zhu, X. Ye, L.Yang, B. Wang, X. Liang, L. Lu, Y. P. Tsao, S. L. Chen, J. Li, X. Xiao,Human gene therapy 21, 109-119 (2010).) The properties ofnon-pathogenicity, broad host range of infectivity, includingnon-dividing cells, and potential site-specific chromosomal integrationmake AAV an attractive tool for gene transfer.

The first rAAV-based gene therapy to be approved in the Western world(Glybera® for lipoprotein lipase deficiency, approved for use in 2012 inthe European Union) has stimulated the gene therapy community, investorsand regulators to the real possibility of moving rAAV therapies into theclinic globally. Yet, despite the impressive abilities of rAAV totransduce a variety of tissue and cell types, human liver tissue hasbeen historically a challenging tissue to transduce at high levelssufficient to provide sustained therapeutic levels of expression ofdelivered transgene products. This likely stems from the fact thatpreclinical modeling with rAAV to determine the best capsid serotypesfor transducing target tissues is done in animal models—typicallymice—which do not necessarily recapitulate the tissue and cell tropismeach rAAV has in humans, nor the transduction capabilities at treatment,as well as the immunological barriers present in humans.

A variety of published US applications describe AAV vectors and virions,including U.S. Publication Nos. 2015/0176027, 2015/0023924,2014/0348794, 2014/0242031, and 2012/0164106; all of which areincorporated by reference herein in their entireties.

However, high levels of transduction are needed for gene therapy trials.If a variant AAV capsid polypeptide exhibited an enhanced neutralizationprofile and/or exhibited increased transduction or tropism in humanliver tissue or hepatocyte cells (i.e., human hepatocyte cells), a lowerdose and fewer injections would be needed to achieve therapeuticrelevance. Similarly, for use as a vaccine delivery tool, highefficiency transduction and stability is needed to achieve robustsecretion of antibodies encoded within the AAV to reach therapeuticlevels of circulating antibodies in the blood.

There remains, therefore, a need in the art for variant AAV capsidswhich exhibit an enhanced neutralization profile, exhibit increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells), or variant AAV capsids that exhibit both. Thepresent invention meets this need by providing variant AAV capsidpolypeptides which demonstrate an enhanced neutralization profile,increased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells), as well as variant AAV capsidsthat exhibit both properties. The present invention utilizes directedevolution by DNA gene shuffling to characterize and screen for suchvariant AAV capsid polypeptides which have an enhanced neutralizationprofile, increased transduction or tropism in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells), as well as variant AAVcapsids that exhibit both properties.

BRIEF SUMMARY OF THE INVENTION

The present invention provides variant adeno-associated virus (AAV)capsid polypeptides, where the variant AAV capsid polypeptides exhibitan enhanced neutralization profile as compared to non-variant parentcapsid polypeptides.

In some embodiments, the variant AAV capsid polypeptides exhibit anenhanced neutralization profile against pooled human immunoglobulins ascompared to non-variant parent capsid polypeptides.

In some embodiments, the variant AAV capsid polypeptides further exhibitincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to non-variant parentcapsid polypeptides.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsid.

In some embodiments, the variant AAV capsid polypeptide further exhibitsincreased transduction or tropism in one or more non-liver human tissuesor non-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vivo as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vitro as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver organoids in 3-dimensionalcultures in vitro as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide is part of afunctional AAV capsid, wherein said functional AAV capsid packages anucleic acid sequence selected from the group consisting of a non-codingRNA, a protein coding sequence, an expression cassette, amulti-expression cassette, a sequence for homologous recombination, agenomic gene targeting cassette, and a therapeutic expression cassette.

In some embodiments, the nucleic acid sequence is contained within anAAV vector.

In some embodiments, the expression cassette is a CRISPR/CAS expressionsystem.

In some embodiments, the therapeutic expression cassette encodes atherapeutic protein or antibody.

In some embodiments, the variant AAV capsid polypeptide allows forenhanced nucleic acid expression as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide sequence isselected from the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59(SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).

The present invention also provides a method of using the variant AAVcapsid polypeptide in a therapeutic treatment regimen or vaccine.

In some embodiments, the method of using the variant AAV capsidpolypeptides of the invention provides for a reduction in the amount oftotal nucleic acid administered to a subject. In some embodiments, themethod comprises administering less total nucleic acid amount to asubject when the nucleic acid is transduced using a variant AAV capsidpolypeptide as compared to the amount of nucleic acid administered to asubject when the nucleic acid is transduced using a non-variant parentcapsid polypeptide in order to obtain a similar therapeutic effect.

The present invention also provides variant adeno-associated virus (AAV)capsid polypeptides, where the variant AAV capsid polypeptides exhibitincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsid.

In some embodiments, the variant AAV capsid polypeptide further exhibitsan enhanced neutralization profile as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibits anenhanced neutralization profile against pooled human immunoglobulins ascompared to a non-variant parent capsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide further exhibitsincreased transduction or tropism in one or more non-liver human tissuesor non-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vivo as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vitro as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver organoids in 3-dimensionalcultures in vitro as compared to a non-variant parent capsid polypeptide

In some embodiments, the variant AAV capsid polypeptide is part of afunctional AAV capsid, wherein said functional AAV capsid packages anucleic acid sequence selected from the group consisting of a non-codingRNA, a protein coding sequence, an expression cassette, amulti-expression cassette, a sequence for homologous recombination, agenomic gene targeting cassette, and a therapeutic expression cassette.

In some embodiments, the nucleic acid sequence is contained within anAAV vector.

In some embodiments, the expression cassette is a CRISPR/CAS expressionsystem.

In some embodiments, the therapeutic expression cassette encodes atherapeutic protein or antibody.

In some embodiments, the variant AAV capsid polypeptide allows forenhanced nucleic acid expression as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide sequence isselected from the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59(SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).

The present invention also provides a method of using the variant AAVcapsid polypeptide in a therapeutic treatment regimen or vaccine.

In some embodiments, the method of using the variant AAV capsidpolypeptide of the invention provides for a reduction in the amount oftotal nucleic acid administered to a subject. In some embodiments, themethod comprises administering less total nucleic acid amount to asubject when the nucleic acid is transduced using a variant AAV capsidpolypeptide as compared to the amount of nucleic acid administered to asubject when the nucleic acid is transduced using a non-variant parentcapsid polypeptide in order to obtain a similar therapeutic effect.

The present invention also provides an adeno-associated virus (AAV)vector comprising a nucleic acid sequence encoding a variant AAV capsidpolypeptide, where the variant AAV capsid polypeptide exhibits anenhanced neutralization profile as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibits anenhanced neutralization profile against pooled human immunoglobulins ascompared to a non-variant parent capsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells).

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide further exhibitsincreased transduction or tropism in one or more non-liver human tissuesor non-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vivo as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vitro as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver organoids in 3-dimensionalcultures in vitro as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the vector further comprises a nucleic acidsequence selected from the group consisting of a non-coding RNA, acoding sequence, an expression cassette, a multi-expression cassette, asequence for homologous recombination, a genomic gene targetingcassette, and a therapeutic expression cassette.

In some embodiments, the variant AAV capsid polypeptide allows forenhanced nucleic acid expression as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the expression cassette is a CRISPR/CAS expressionsystem.

In some embodiments, the therapeutic expression cassette encodes atherapeutic protein or antibody.

In some embodiments, the variant AAV capsid polypeptide sequence isselected from the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59(SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).

The present invention also provides a method of using the variant AAVcapsid polypeptide in a therapeutic treatment regimen or vaccine.

The present invention also provides an adeno-associated virus (AAV)vector comprising a nucleic acid sequence encoding a variant AAV capsidpolypeptide, where the variant AAV capsid polypeptide exhibits increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsid.

In some embodiments, the variant AAV capsid polypeptide further exhibitsan enhanced neutralization profile as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibits anenhanced neutralization profile against pooled human immunoglobulins ascompared to a non-variant parent capsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide further exhibitsincreased transduction or tropism in one or more non-liver human tissuesor non-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vivo as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vitro as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver organoids in 3-dimensionalcultures in vitro as compared to a non-variant parent capsid polypeptide

In some embodiments, the vector further comprises a nucleic acidsequence selected from the group consisting of a non-coding RNA, acoding sequence, an expression cassette, a multi-expression cassette, asequence for homologous recombination, a genomic gene targetingcassette, and a therapeutic expression cassette.

In some embodiments, the variant AAV capsid polypeptide allows forenhanced nucleic acid expression as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the said expression cassette is a CRISPR/CASexpression system.

In some embodiments, the said therapeutic expression cassette encodes atherapeutic protein or antibody.

In some embodiments, the variant AAV capsid polypeptide sequence isselected from the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59(SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8). Insome embodiments, the variant AAV capsid polypeptide is selected fromthe group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ ID NO:4),and AAV-NP40 (SEQ ID NO:6). In some embodiments, the variant AAV capsidpolypeptide is selected from the group consisting of AAV-NP59 (SEQ IDNO:4), and AAV-NP40 (SEQ ID NO:6). In some embodiments, the variant AAVcapsid polypeptide is AAV-NP59 (SEQ ID NO:4). In some embodiments, thevariant AAV capsid polypeptide is AAV-NP40 (SEQ ID NO:6).

The present invention also provides a method of using the variant AAVcapsid polypeptide in a therapeutic treatment regimen or vaccine.

In some embodiments, the method of using the AAV vector of the inventionprovides for a reduction in the amount of total AAV vector administeredto a subject. In some embodiments, the method comprises administeringless total AAV vector amount to said subject when said AAV vector istransduced by a variant AAV capsid polypeptide as compared to the amountof AAV vector administered to said subject when said AAV vector istransduced by a non-variant parent capsid polypeptide in order to obtaina similar therapeutic effect.

The present invention also provides methods for generating variant AAVcapsid polypeptides, wherein the variant AAV capsid polypeptide exhibitsboth an enhanced neutralization profile and increased transduction ortropism in human liver tissue or hepatocyte cells (i.e., humanhepatocyte cells) as compared to a non-variant parent capsidpolypeptide. In some embodiments, the method comprises:

-   -   a) generating a library of variant AAV capsid polypeptides,        wherein said variant AAV capsid polypeptides include a plurality        of variant AAV capsid polypeptide sequences from more than one        non-variant parent capsid polypeptide;    -   b) generating an AAV vector library by cloning said variant AAV        capsid polypeptide library into AAV vectors, wherein said AAV        vectors are replication competent AAV vectors;    -   c) screening said AAV vector library from b) for variant AAV        capsid polypeptides for both an enhanced neutralization profile        and increased transduction or tropism in human liver tissue or        hepatocyte cells (i.e., human hepatocyte cells) as compared to a        non-variant parent capsid polypeptide; and    -   d) selecting said variant AAV capsid polypeptides from c).

In some embodiments, the method further comprises e) determining thesequence of said variant AAV capsid polypeptides from d).

In some embodiments, the variant AAV capsid polypeptide exhibits anenhanced neutralization profile against pooled human immunoglobulins ascompared to a non-variant parent capsid polypeptide

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide further exhibitsincreased transduction or tropism in one or more non-liver human tissuesor non-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vivo as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in vitro as compared to a non-variant parentcapsid polypeptide.

In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human liver organoids in 3-dimensionalcultures in vitro as compared to a non-variant parent capsid polypeptide

In some embodiments, the variant AAV capsid polypeptide exhibitsselective transduction of human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) in chimeric mice having undergone xenografttransplants with human liver tissue or hepatocyte cells (i.e., humanhepatocyte cells).

Other objects, advantages and embodiments of the invention will beapparent from the detailed description following.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides confocal immunofluorescence (IF) staining highlightingthe species preference of AAV serotypes DJ and LK03 for mouse and humanhepatocytes, respectively.

FIG. 2A-FIG. 2D provides a schematic regarding the directed evolution ofadeno-associated virus capsids by DNA shuffling and multiplexedsequential screening in humanized liver mice and against pooled humanimmunoglobulins. (A) AAV capsid genes from ten parental serotypes (1, 2,3b, 4, 5, 6, 8, 9_hu14, avian and bovine) were PCR-amplified, fragmentedwith DNaseI digestion and then randomly reassembled through self-primingPCR. Resultant shuffled Cap genes were cloned back into areplication-competent AAV production plasmid via flanking SwaI/NsiIsites downstream of AAV2 Rep. The resultant library production plasmidcontained AAV2 ITRs and a modified AAV2 3′UTR. The AAV library waspackaged using standard production protocols, dot blot titered and usedfor selection. (B) Diagram illustrating the procedure for producinghumanized liver FRG mice with NTBC selection for selection in humanizedliver mice. (C) Diagram illustrating the initial 5-round selectionscreen with replicating AAV capsid libraries from (A) in humanized livermice from (B). (D) Diagram illustrating the subsequent 2-round subscreenagainst pooled human immunoglobulins with the evolved AAV libraryalready screened for human hepatocyte tropism. Capsids which did notbind pooled human immunoglobulins were taken for furthercharacterization.

FIG. 3 provides a schematic showing the multiplexed sequential AAVcapsid screens for human liver tropism and reduced human immunoglobulin(IgG) binding.

FIG. 4 provides a schematic showing the sequential sub-screen on round 5selected AAV capsids for reduced humoral neutralization by pooled humanIgGs.

FIG. 5 provides a diagram showing the phylogeny of 100 selected variantsand illustrates “families” of related capsids which evolved from thesequential screens.

FIG. 6 provides data regarding IVIG binding assays on pools of closelyrelated AAV chimeras.

FIG. 7 provides data regarding capsid enrichment scores for 4 variantsfrom Pool A (AAV-NP59 (SEQ ID NO:4), AAV-NP84 (SEQ ID NO:2), AAV-NP40(SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8)).

FIG. 8 provides a diagram showing parental contribution to chimericcapsid composition of Pool A variants from the library screens.

FIG. 9 provides confocal immunofluorescence (IF) staining of humanhepatocytes in chimeric mice transplanted with human hepatocytes invivo. Column 1: Hoechst stained nuclei (blue). Column 2: humanhepatocyte staining of human FAH protein (red). Column 3: AAV transducedcells expressing green fluorescent protein (GFP) (green). Column 4:overlay of columns 1, 2, and 3.

FIG. 10 provides data regarding neutralization assays on human HEK293and human Huh7 cells with each AAV in the presence of increasingconcentrations of pooled human immunoglobulins (IVIG) (AAV-NP59 (SEQ IDNO:4), AAV-NP84 (SEQ ID NO:2), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQID NO:8)). The IVIG neutralization assay was adopted from severalpreviously described methods (Grimm, et al., J. Virol. 82(12): 5887-5911(2008); Arbetman, et al. J. Virol. 79(24):15238-15245 (2005)), withmodifications. Gammagard IVIG Liquid [100 mg/mL] (Baxter, ProductCode#LE1500190, Lot#LE12P180AB) was used with a concentration range of0-50 mg/mL (0, 0.1, 1, 2.5, 5, 10, 25, 50 mg/mL). ssAAV-CAG-GFP was usedas the transfer vector. An identical number of genome-containing vectorparticles (MOI 75,000) of each variant were incubated with increasingconcentrations of IVIG at 37° C. for 1-hr. During this hour, the cellculture media on both cell types was changed to that lacking any serumor antibiotics/antimycotics. 80,000 cells (either HEK293 or Huh7) weretransduced with the IVIG/AAV mixture and cultures were washed 6-hr laterand cultured for 72-hr to allow AAV trafficking, uncoating and GFPexpression. Cells were then harvested and analyzed for GFP expression byFACS. Each serotype was normalized to its own “0 mg/mL IVIG” controlsample and the concentration of IVIG needed to decrease the signal by50% (hence why the X and Y axis on the graph cross at 50% instead of 0%)was determined. 10,000 cells were used for each condition to determinethe counts/statistics.

FIG. 11A-FIG. 11D provides the nucleic acid and amino acid sequences forfour AAV capsids: A) AAV-NP84, B) AAV-NP59, C) AAV-NP40, and D)AAV-NP30.

FIG. 12 provides percent parental conservation at each amino acidposition during the progression of each screen. Using PacBiosingle-molecule sequencing and bioinformatic analyses, positionalenrichment assessments were performed to calculate percent conservationamong amino acids from parental serotypes (AAVs 1, 2, 3b, 4, 5, 6, 8,9_hu14) or de novo mutations for each amino acid position among allcapsids at key rounds during the screen (input, rounds 1 and 5 in vivoand round 7 unbound). Bovine and avian were removed from the plot sincefew variants showed any appreciable contribution from those twoserotypes. The maximum square size indicates that 100% of variants sharethat amino acid from that parent at that position. All other squaresizes are proportional to the percent of variants from 0-100% that havethat amino acid at that position from that parent. Each parent iscolored as is shown in the legend (same color scheme is used in FIG. 3A,B) and de novo mutations that evolved during the screen are shown inblack. VP1, VP2, VP3 and AAP ORFs are diagrammed below for reference.

FIG. 13A-FIG. 13B provides sequence and structural composition of newhuman hepatotropic shuffled AAV capsid variants. (A) Crossover mappinganalysis of capsid fragment crossovers in vectorized variants from theparental AAV serotypes (AAVs 1, 2, 3b, 4, 5, 6, 8, 9_hu14) used in thelibrary. The maximum circle size indicates a 100% match for that aminoacid from that parent at that position. All other circle sizes areproportional to the percent likelihood that that amino acid at thatposition matches that parent. The solid black line for each chimerarepresents the most likely parental serotype match identified acrosseach crossover. Thin parallel lines between crossovers indicate multipleparental matches at an equal probability. Vertical spikes indicate amutation from within the parental sequence space, while an overheadasterisk indicates an evolved de novo mutation for which no parent hasthat amino acid at that position. VP1, VP2, VP3 and AAP ORFs arediagrammed below for reference. (B) Shuffled variants were 3Dfalse-color mapped onto the crystal structure of AAV2. Color-codingindicates parental contribution using the same colors as in (A).

FIG. 14A-FIG. 14C provides validation and comparative quantitation ofhuman hepatocyte transduction in humanized liver mice in vivo. (A)Representative immunohistochemical images from treated humanized livermice from OHSU transduced with ssAAV-CAG-GFP at 2E11 vg IV with varyingcapsid serotypes. Human-specific FAH (violet), viral-GFP (green),Hoechst (blue) on liver cross-sections. Scale=100-μM. (B) Representativeimmunohistochemical images from treated humanized liver mice from CMRItransduced with ssAAV-LSP1-GFP at 2E11 vg IV with varying capsidserotypes. Human-specific albumin (red), viral-GFP (green), DAPI (blue)on liver cross-sections. Scale=100 μM. (C) Summary of analysis fromtransduced xenografted mice. The percentage of transduced humanhepatocytes was determined by individually analyzing and comparing cellcounts from images of GFP fluorescence and human FAH immunostaining asdescribed previously⁴.

FIG. 15A-FIG. 15D provides immunological assays across key patientgroups and nonhuman primates. (A) Seroreactivity ELISA assay forpresence of anti-AAV antibodies in normal human serum from 50 U.S.adults. Each patient was assayed in technical triplicates with datapoints representing the mean minus background. Red line represents themean. Symbols are consistent across treatments for each patient to allowcomparisons. (B) Seroreactivity ELISA assay for presence of anti-AAVantibodies in serum from 6 rhesus macaques. Each dot is 1 macaque. Redline represents the mean. (C) Human 2V6.11 AAV-permissive cells wereused to assess rAAV neutralization in the presence of patient serums for21 E.U. individuals. Data show the reciprocal dilution at which >50%inhibition of transduction was observed. Red line represents the mean.(D) Seroreactivity ELISA assay for presence of anti-AAV antibodies inhuman serum from 21 adult males with hemophilia B. Red line representsthe mean.

FIG. 16A-FIG. 16B provides comparative ex vivo human liver organoidtransduction with AAV. (A) Transduction assessments by GFP expression ofvectorized variants in primary human liver organoid cultures compared toknown hepatotropic AAV control serotypes 2, 8, DJ and LK03. Eachserotype was assayed in a 14-day time-course in technical duplicates;day 11 shown. Scale=100-μM. Media from organoid cultures was tested forthe presence of human albumin by ELISA (human albumin=58.3-ng/mL). (B)GFP FACS quantitation data on 100,000 dissociated organoid cells from(A) at the end of the study at day 14.

FIG. 17A-FIG. 17E provides data regarding the lack of functional humanhepatocyte transduction with AAV-DJ. (A) Representative staining fromxenografted FRG livers administered ssAAV-DJ-CAG-GFP at 2E11 vg IV.Sections were stained with Hoechst (blue), viral GFP (green), andhuman-specific FAH (violet). Scale=100-μM. (B) Summary of analysis fromtransduced xenografted mice. (C) GFP RNA FISH and GFP protein IF overlayon sections from (A). Human and mouse hepatocytes are differentiated byDAPI labeling (mouse nuclei=large with bright blue heterochromaticpunctae (green arrows); human nuclei=small and dull diffuse blue (whitearrows)). GFP RNA FISH probed for the AAV-GFP genome (red). Green GFPprotein immunofluorescence was then overlaid on top of the GFP RNA probeand image coordinates were recorded. Few human hepatocytes showed DJ-GFPRNA, but none showed concomitant GFP protein expression. Many mousehepatocytes showed DJ-GFP RNA, and also had concomitant GFP proteinexpression. Scale=10-μM. (D) Subsequent GFP DNA FISH (red) after RNAFISH on liver sections from (b). Rare human hepatocytes showednucleoplasmic concatemers of AAV dsDNA (red punctate dots, pink arrow).This cumulatively showed successful AAV uncoating, dsDNA conversion, andin some cases even transcription into RNA, but a block at translatingthose genomes into GFP protein. (E) In comparison, AAV-LK03-GFP positivecontrol RNA FISH showed abundant cytoplasmic and nucleolar GFP RNA(red). RNAse A treatment followed by GFP RNA FISH eliminated signalsseen with RNA FISH alone. Sequential GFP DNA FISH done after RNAse A andRNA FISH showed presence of bright nucleoplasmic GFP DNA signal,confirming that the signal from RNA FISH was ssRNA and not mis-probedssDNA AAV genome signal.

FIG. 18 provides the modified AAV2 VP1 3′UTR sequence in library cloningplasmid. A modified 3′-UTR sequence from AAV2 was maintained during thecloning of the recipient library plasmid to ensure proper expression andreplication of AAV genomes.

FIG. 19A-FIG. 19B provides the phylogenetic trees of screen progressutilizing PacBio full-length capsid sequencing. (A) Comparativephylogenies showing genetic relatedness at the amino acid level amongthe parental serotypes in the library and all library variants. Thedecreasing diversity and increasing enrichment going from the unselectedAAV library through 5 rounds of in vivo selection and 2 rounds ofon-bead IgG selection are shown. (B) Raw and filtered CCS read countsthat were used to generate (a) are shown.

FIG. 20A-FIG. 20B provides phylogeny and enrichment scores of topvariants from screen completion. (A) Phylogenetic tree showing geneticrelatedness at the amino acid level among the parental serotypes in thelibrary (AAVs 1, 2, 3b, 4, 5, 6, 8, 9_hu14) and the top 100 selectedvariants from the final round of the second screen for immune evasionfrom the unbound AAV fraction. (B) Enrichment scores were calculated foreach amino acid position in the sequence of each chimera by comparisonof sequences from parental serotypes based on maximum likelihood.Library parents are depicted in different colors as shown.

FIG. 21A-FIG. 21B provides short in vivo transduction time-course innon-humanized mice. (A) Live imaging FLuc transduction time course innon-transplanted wildtype Balb/cJ mice treated with PBS, AAV-DJ, AAV8,AAV-NP30 (SEQ ID NO:8), AAV-NP40 (SEQ ID NO;6), AAV-NP59 (SEQ ID NO:4)or AAV-NP84 (SEQ ID NO:2) expressing ssAAV-EF1α-FLuc after IV tail veinadministration (2E11 vg/mouse). Mean radiance (p/s/cm2/sr) is displayedwith all mice imaged on the same, shared scale. All mice were imaged onboth the dorsal (d) and ventral (v) sides. (B) Quantified dorsal andventral radiance at day 14 from mice from (A).

FIG. 22A-FIG. 22B provides seroreactivity ELISA profiling separated bygender. Breakdown of human seroreactivity from FIG. 5a by gender.Symbols are consistent across treatments for each patient to allowcomparisons. (A) Male patients. (B) Female patients. Line represents themean.

FIG. 23A-FIG. 23C provides the amino acid sequences of best performingshuffled capsid variants.

FIG. 24 provides data regarding the transduction of human hepatocytes inthe humanized mice for each of 4 vectors (NP59, NP40, NP84, and LK03) ata dose 5×10¹⁰ vg.

DETAILED DESCRIPTION OF THE INVENTION Introduction

There remains a need in the art for gene therapy vectors capable ofexhibiting enhanced neutralization profiles as well as vectors withincreased transduction and/or tropism in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells) for gene therapy, sothat more therapeutic levels of nucleic acid expression can be achieved.The present invention meets this need and provides variant AAV capsidpolypeptides which exhibit an enhanced neutralization profile ascompared to non-variant parent capsid polypeptides. The presentinvention also meets this need and provides variant AAV capsidpolypeptides which exhibit increased transduction and/or tropism inhuman liver tissue or hepatocyte cells (i.e., human hepatocyte cells) ascompared to non-variant parent capsid polypeptides. The presentinvention additionally meets this need by providing variant AAV capsidpolypeptides which exhibit both an enhanced neutralization profile ascompared to non-variant parent capsid polypeptides and exhibit increasedtransduction and/or tropism in human liver tissue or hepatocyte cells(i.e., human hepatocyte cells) as compared to non-variant parent capsidpolypeptides.

Detailed Description

In various embodiments, the present invention provides variantadeno-associated virus (AAV) capsid polypeptides (i.e., variant AAVcapsid polypeptides), where the variant AAV capsid polypeptides exhibitan enhanced neutralization profile as compared to non-variant parentcapsid polypeptides. In some embodiments, the variant AAV capsidpolypeptides exhibit an enhanced neutralization profile against pooledhuman immunoglobulins as compared to a non-variant parent capsidpolypeptide. In some embodiments, the variant AAV capsid polypeptidefurther exhibits increased transduction or tropism in human liver tissueor hepatocyte cells (i.e., human hepatocyte cells) as compared to anon-variant parent capsid polypeptide.

In various embodiments, the present invention provides variantadeno-associated virus (AAV) capsid polypeptides, wherein the variantcapsid polypeptides exhibit increased transduction or tropism in humanliver tissue or hepatocyte cells (i.e., human hepatocyte cells) ascompared to non-variant parent capsid polypeptides. In some embodiments,the variant AAV capsid polypeptide further exhibits an enhancedneutralization profile against pooled human immunoglobulins as comparedto a non-variant parent capsid polypeptide.

In some embodiments the variant AAV capsid polypeptide is referred to asa variant recombinant AAV capsid polypeptide or variant rAAV capsidpolypeptide.

In other various embodiments, the present invention provides AAV vectorscomprising a nucleic acid sequence coding for a variant capsidpolypeptide, where the variant AAV capsid polypeptides exhibit anenhanced neutralization profile as compared to non-variant parent capsidpolypeptides. In some embodiments, the variant AAV capsid polypeptidesexhibit an enhanced neutralization profile against pooled humanimmunoglobulins as compared to a non-variant parent capsid polypeptide.In some embodiments the AAV vector is referred to as a recombinant AAVor rAAV vector.

In other various embodiments, the present invention provides AAV vectorscomprising a nucleic acid sequence coding for a variant capsidpolypeptide, wherein the variant capsid polypeptide exhibits increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) as compared to a vector encoding a non-variantparent capsid polypeptide. In some embodiments the AAV vector isreferred to as a recombinant AAV or rAAV vector.

In other various embodiments, the present invention provides AAV vectorscomprising a nucleic acid sequence coding for a variant capsidpolypeptide, wherein the variant capsid polypeptide exhibits bothincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a vector encoding anon-variant parent capsid polypeptide and an enhanced neutralizationprofile as compared to non-variant parent capsid polypeptides. In someembodiments, the variant AAV capsid polypeptides exhibit an enhancedneutralization profile against pooled human immunoglobulins as comparedto a non-variant parent capsid polypeptide. In some embodiments the AAVvector is referred to as a recombinant AAV or rAAV vector.

In some embodiments, the present invention provides AAV vectorscomprising: a) a variant AAV capsid protein, wherein the variant AAVcapsid polypeptide comprises at least one amino acid difference (e.g.,amino acid substitution, amino acid insertion, amino acid deletion)relative to a substantially identical non-variant parent AAV capsidprotein, and where the variant capsid protein exhibits an enhancedneutralization profile as compared to a vector encoding a non-variantparent capsid polypeptide. In some embodiments, the AAV capsidpolypeptide does not comprise an amino acid sequence present in anaturally occurring AAV capsid polypeptide.

In some embodiments, the present invention provides AAV vectorscomprising: a) a variant AAV capsid protein, wherein the variant AAVcapsid polypeptide comprises at least one amino acid difference (e.g.,amino acid substitution, amino acid insertion, amino acid deletion)relative to a substantially identical non-variant parent AAV capsidprotein, and where the variant capsid protein exhibits increasedtransduction or tropism in liver tissue or hepatocyte cells as comparedto a vector encoding a non-variant parent capsid polypeptide. In someembodiments, the AAV capsid polypeptide does not comprise an amino acidsequence present in a naturally occurring AAV capsid polypeptide.

In some embodiments, the present invention provides AAV vectorscomprising: a) a variant AAV capsid protein, wherein the variant AAVcapsid polypeptide comprises at least one amino acid difference (e.g.,amino acid substitution, amino acid insertion, amino acid deletion)relative to a substantially identical non-variant parent AAV capsidprotein, and where the variant capsid protein exhibits both increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) as compared to a vector encoding a non-variantparent capsid polypeptide and an enhanced neutralization profile ascompared to a vector encoding a non-variant capsid polypeptide. In someembodiments, the AAV capsid polypeptide does not comprise an amino acidsequence present in a naturally occurring AAV capsid polypeptide.

In some embodiments, the present invention provides variant AAV capsidpolypeptides, wherein the variant AAV capsid polypeptide comprises atleast one amino acid difference (e.g., amino acid substitution, aminoacid insertion, amino acid deletion) relative to a substantiallyidentical non-variant parent AAV capsid polypeptide, and where thevariant capsid polypeptide exhibits an enhanced neutralization profileas compared to a vector encoding a non-variant parent capsidpolypeptide. In some embodiments, the variant AAV capsid polypeptidedoes not comprise an amino acid sequence present in a naturallyoccurring AAV capsid polypeptide.

In some embodiments, the present invention provides variant AAV capsidpolypeptides, wherein the variant AAV capsid polypeptide comprises atleast one amino acid difference (e.g., amino acid substitution, aminoacid insertion, amino acid deletion) relative to a substantiallyidentical non-variant parent AAV capsid protein, and where the variantcapsid polypeptide exhibits increased transduction or tropism in humanliver tissue or hepatocyte cells (i.e., human hepatocyte cells) ascompared to a vector encoding a non-variant parent capsid polypeptide.In some embodiments, the variant AAV capsid polypeptide does notcomprise an amino acid sequence present in a naturally occurring AAVcapsid polypeptide.

In some embodiments, the present invention provides variant AAV capsidpolypeptides, wherein the variant AAV capsid polypeptide comprises atleast one amino acid difference (e.g., amino acid substitution, aminoacid insertion, amino acid deletion) relative to a substantiallyidentical non-variant parent AAV capsid polypeptide, and where thevariant capsid polypeptide exhibits both an enhanced neutralizationprofile as compared to a vector encoding a non-variant parent capsidpolypeptide and increased transduction or tropism in human liver tissueor hepatocyte cells (i.e., human hepatocyte cells) as compared to avector encoding a non-variant parent capsid polypeptide. In someembodiments, the variant AAV capsid polypeptide does not comprise anamino acid sequence present in a naturally occurring AAV capsidpolypeptide.

Before the invention is described in greater detail, it is to beunderstood that the invention is not limited to particular embodimentsdescribed herein as such embodiments may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context presented, provides the substantialequivalent of the specifically recited number. All publications,patents, and patent applications cited in this specification areincorporated herein by reference to the same extent as if eachindividual publication, patent, or patent application were specificallyand individually indicated to be incorporated by reference. Furthermore,each cited publication, patent, or patent application is incorporatedherein by reference to disclose and describe the subject matter inconnection with which the publications are cited. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the invention described herein isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided might be different from theactual publication dates, which may need to be independently confirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featuresreadily separated from or combined with the features of any of the otherseveral embodiments without departing from the scope or spirit of theinvention. Any recited method may be carried out in the order of eventsrecited or in any other order that is logically possible. Although anymethods and materials similar or equivalent to those described hereinmay also be used in the practice or testing of the invention,representative illustrative methods and materials are now described.

As described in the present invention, the following terms will beemployed, and are defined as indicated below.

Abbreviations

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”).

Definitions

The term “AAV” includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3(AAV3), AAV type 3b (AAV3b), AAV type 4 (AAV4), AAV type 5 (AAV5), AAVtype 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9),AAV 9_hu14, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV capable ofinfecting primates, “non-primate AAV” refers to AAV capable of infectingnon-primate mammals, “bovine AAV” refers to AAV capable of infectingbovine mammals, etc.

An “AAV vector” as used herein refers to an AAV vector nucleic acidsequence encoding for various nucleic acid sequences, including in someembodiments a variant capsid polypeptide (i.e., the AAV vector comprisesa nucleic acid sequence encoding for a variant capsid polypeptide). Thevariant capsid polypeptide exhibits an enhanced neutralization profile,an increased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) or, in some embodiments, both, ascompared to a non-variant parent capsid polypeptide. The AAV vectors canalso comprise a heterologous nucleic acid sequence not of AAV origin aspart of the nucleic acid insert. This heterologous nucleic acid sequencetypically comprises a sequence of interest for the genetictransformation of a cell. In general, the heterologous nucleic acidsequence is flanked by at least one, and generally by two AAV invertedterminal repeat sequences (ITRs).

The phrase “non-variant parent capsid polypeptide” includes anynaturally occurring AAV capsid polypeptide and/or any AAV wild-typecapsid polypeptide. In some embodiments, the non-variant parent capsidpolypeptide includes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV8,AAV9_hu14, bovine AAV and/or avian AAV capsid polypeptides. In someembodiments, the non-variant parent capsid polypeptide can also be acontrol capsid polypeptide, including for example but not limited toLK03 and/or DJ, as described herein and known in the art. In someembodiments, a control capsid polypeptide is LK03. In some embodiments,a control capsid polypeptide is DJ.

The term “substantially identical” in the context of variant capsidpolypeptides and non-variant parent capsid polypeptides refers tosequences with 1 or more amino acid changes. In some embodiments, thesechanges do not affect the packaging function of the capsid polypeptide.In some embodiments, substantially identical include variant capsidpolypeptides about 99%, about 98%, about 97%, about 96%, about 95%,about 94%, about 93%, about 92%, about 91%, or about 90% identical to anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide can be substantially identical to a non-variantparent capsid polypeptide over a subregion of the variant capsidpolypeptide, such as over about 25%, about 50%, about 75%, or about 90%of the total polypeptide sequence length.

An “AAV virion” or “AAV virus” or “AAV viral particle” or “AAV vectorparticle” refers to a viral particle composed of at least one AAV capsidpolypeptide (including both variant capsid polypeptides and non-variantparent capsid polypeptides) and an encapsidated polynucleotide AAVtransfer vector. If the particle comprises a heterologous nucleic acid(i.e. a polynucleotide other than a wild-type AAV genome, such as atransgene to be delivered to a mammalian cell), it can be referred to asan “AAV vector particle” or simply an “AAV vector”. Thus, production ofAAV virion or AAV particle necessarily includes production of an AAVvector as such a vector is contained within an AAV virion or an AAVparticle.

“Packaging” refers to a series of intracellular events resulting in theassembly of AAV virions or AAV particles which encapsidate a nucleicacid sequence and/or other therapeutic molecule. Packaging can refer toencapsidation of nucleic acid sequence and/or other therapeuticmolecules into a capsid comprising the variant capsid polypeptidesdescribed herein.

The phrase “therapeutic molecule” as used herein can include nucleicacids (including, for example, vectors), polypeptides (including, forexample, antibodies), and vaccines, as well as any other therapeuticmolecule that could be packaged by the variant AAV capsid polypeptidesof the invention.

AAV “rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus (AAV).AAV Rep (replication) and Cap (capsid) are referred to herein as AAV“packaging genes.”

A “helper virus” for AAV refers to a virus allowing AAV (e.g. wild-typeAAV) to be replicated and packaged by a mammalian cell. A variety ofsuch helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used as a helper virus.Numerous adenoviruses of human, non-human mammalian and avian origin areknown and available from depositories such as the ATCC. Viruses of theherpes family include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome allowing AAV replication and packaging (in conjunction withother requirements for replication and packaging described herein). Asdescribed herein, “helper virus function” may be provided in a number ofways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans.

An “infectious” virion, virus or viral particle is one comprising apolynucleotide component deliverable into a cell tropic for the viralspecies. The term does not necessarily imply any replication capacity ofthe virus. As used herein, an “infectious” virus or viral particle isone that upon accessing a target cell, can infect a target cell, and canexpress a heterologous nucleic acid in a target cell. Thus,“infectivity” refers to the ability of a viral particle to access atarget cell, infect a target cell, and express a heterologous nucleicacid in a target cell. Infectivity can refer to in vitro infectivity orin vivo infectivity. Assays for counting infectious viral particles aredescribed elsewhere in this disclosure and are known in the art. Viralinfectivity can be expressed as the ratio of infectious viral particlesto total viral particles. Total viral particles can be expressed as thenumber of viral genome copies. The ability of a viral particle toexpress a heterologous nucleic acid in a cell can be referred to as“transduction.” The ability of a viral particle to express aheterologous nucleic acid in a cell can be assayed using a number oftechniques, including assessment of a marker gene, such as a greenfluorescent protein (GFP) assay (e.g., where the virus comprises anucleotide sequence encoding GFP), where GFP is produced in a cellinfected with the viral particle and is detected and/or measured; or themeasurement of a produced protein, for example by an enzyme-linkedimmunosorbent assay (ELISA) or fluorescence-activated cell sorting(FACS).

A “replication-competent” virion or virus (e.g. a replication-competentAAV) refers to an infectious phenotypically wild-type virus, and isreplicable in an infected cell (i.e. in the presence of a helper virusor helper virus functions). In the case of AAV, replication competencegenerally requires the presence of functional AAV packaging genes. Insome embodiments, AAV vectors, as described herein, lack of one or moreAAV packaging genes and are replication-incompetent in mammalian cells(especially in human cells). In some embodiments, AAV vectors lack anyAAV packaging gene sequences, minimizing the possibility of generatingreplication-competent AAV by recombination between AAV packaging genesand an incoming AAV vector. In many embodiments, AAV vector preparationsas described herein are those containing few if anyreplication-competent AAV (rcAAV, also referred to as RCA) (e.g., lessthan about 1 rcAAV per 10² AAV particles, less than about 1 rcAAV per10⁴ AAV particles, less than about 1 rcAAV per 10⁸ AAV particles, lessthan about 1 rcAAV per 10¹² AAV particles, or no rcAAV).

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein to refer to all forms of nucleic acid, oligonucleotides,including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Polynucleotides include genomic DNA, cDNA and antisense DNA, and splicedor unspliced mRNA, rRNA, tRNA, lncRNA, RNA antagomirs, and inhibitoryDNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA(miRNA), aptamers, small or short interfering (si)RNA, trans-splicingRNA, or antisense RNA). Polynucleotides also include non-coding RNA,which include for example, but are not limited to, RNAi, miRNAs,lncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs knownto those of skill in the art. Polynucleotides include naturallyoccurring, synthetic, and intentionally altered or modifiedpolynucleotides as well as analogues and derivatives. The term“polynucleotide” also refers to a polymeric form of nucleotides of anylength, including deoxyribonucleotides or ribonucleotides, or analogsthereof, and is synonymous with nucleic acid sequence. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andnucleotide analogs, and may be interrupted by non-nucleotide components.If present, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The term polynucleotide, asused herein, refers interchangeably to double- and single-strandedmolecules. Unless otherwise specified or required, any embodiment asdescribed herein encompassing a polynucleotide encompasses both thedouble-stranded form and each of two complementary single-stranded formsknown or predicted to make up the double-stranded form. Polynucleotidescan be single, double, or triplex, linear or circular, and can be of anylength. In discussing polynucleotides, a sequence or structure of aparticular polynucleotide may be described herein according to theconvention of providing the sequence in the 5′ to 3′ direction.

A “gene” refers to a polynucleotide containing at least one open readingframe capable of encoding a particular protein or polypeptide afterbeing transcribed and translated.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides targeted to a gene interest (a “target gene”). An“RNA duplex” refers to the structure formed by the complementary pairingbetween two regions of a RNA molecule. siRNA is “targeted” to a gene andthe nucleotide sequence of the duplex portion of the siRNA iscomplementary to a nucleotide sequence of the targeted gene. In someembodiments, the length of the duplex of siRNAs is less than 30nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotidesin length. In some embodiments, the length of the duplex is 19-25nucleotides in length. The RNA duplex portion of the siRNA can be partof a hairpin structure. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences forming the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length.

As used herein, the term “microRNA” refers to any type of interferingRNAs, including but not limited to, endogenous microRNAs and artificialmicroRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAsnaturally encoded in the genome capable of modulating the productiveutilization of mRNA. An artificial microRNA can be any type of RNAsequence, other than endogenous microRNA, capable of modulating theactivity of an mRNA. A microRNA sequence can be an RNA molecule composedof any one or more of these sequences. MicroRNA (or “miRNA”) sequenceshave been described in publications such as Lim, et al., 2003, Genes &Development, 17, 991-1008, Lim et al., 2003, Science, 299, 1540, Lee andAmbrose, 2001, Science, 294, 862, Lau et al., 2001, Science 294,858-861, Lagos-Quintana et al., 2002, Current Biology, 12, 735-739,Lagos-Quintana et al., 2001, Science, 294, 853-857, and Lagos-Quintanaet al., 2003, RNA, 9, 175-179. Examples of microRNAs include any RNAfragment of a larger RNA, or a miRNA, siRNA, stRNA, sncRNA, tncRNA,snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNA. See, e.g.,US Patent Applications 20050272923, 20050266552, 20050142581, and20050075492. A “microRNA precursor” (or “pre-miRNA”) refers to a nucleicacid having a stem-loop structure with a microRNA sequence incorporatedtherein. A “mature microRNA” (or “mature miRNA”) includes a microRNAcleaved from a microRNA precursor (a “pre-miRNA”), or synthesized (e.g.,synthesized in a laboratory by cell-free synthesis), and has a length offrom about 19 nucleotides to about 27 nucleotides, e.g., a maturemicroRNA can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt,25 nt, 26 nt, or 27 nt. A mature microRNA can bind to a target mRNA andinhibit translation of the target mRNA.

“Recombinant,” as applied to a polynucleotide means the polynucleotideis the product of various combinations of cloning, restriction orligation steps, and other procedures resulting in a construct distinctand/or different from a polynucleotide found in nature. A recombinantvirus is a viral particle encapsidating a recombinant polynucleotide.The terms respectively include replicates of the original polynucleotideconstruct and progeny of the original virus construct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules contributing to the functionalregulation of a polynucleotide, including replication, duplication,transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

“Heterologous” means derived from a genotypically distinct entity fromthe rest of the entity it is being compared too. For example, apolynucleotide introduced by genetic engineering techniques into aplasmid or vector derived from a different species is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence it is not naturally found linkedto a heterologous promoter. For example, an AAV including a heterologousnucleic acid encoding a heterologous gene product is an AAV including anucleic acid not normally included in a naturally-occurring, wild-typeAAV, and the encoded heterologous gene product is a gene product notnormally encoded by a naturally-occurring, wild-type AAV. An AAVincluding a nucleic acid encoding a variant capsid polypeptide includesa heterologous nucleic acid sequence. Once transferred/delivered into ahost cell, a heterologous polynucleotide, contained within the virion,can be expressed (e.g., transcribed, and translated, if appropriate).Alternatively, a transferred/delivered heterologous polynucleotide intoa host cell, contained within the virion, need not be expressed.Although the term “heterologous” is not always used herein in referenceto polynucleotides, reference to a polynucleotide even in the absence ofthe modifier “heterologous” is intended to include heterologouspolynucleotides in spite of the omission.

The terms “genetic alteration” and “genetic modification” (andgrammatical variants thereof) are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationsmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate transfection, or contact witha polynucleotide-liposome complex. Genetic alterations may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration changingthe phenotype and/or genotype of the cell and its progeny is included inthis term.

A cell is said to be “stably” altered, transduced, genetically modified,or transformed with a genetic sequence if the sequence is available toperform its function during extended culture of the cell in vitro.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced and inheritable by progeny ofthe altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The “polypeptides,” “proteins” and “peptides” encoded by the“polynucleotide sequences,” include full-length native sequences, aswith naturally occurring proteins, as well as functional subsequences,modified forms or sequence variants so long as the subsequence, modifiedform, or variant retains some degree of functionality of the nativefull-length protein. In methods and uses of as described herein, suchpolypeptides, proteins, and peptides encoded by the polynucleotidesequences can be but are not required to be identical to the defectiveendogenous protein, or whose expression is insufficient, or deficient inthe treated mammal. The terms also encompass a modified amino acidpolymer; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, methylation, carboxylation, deamidation,acetylation, or conjugation with a labeling component. Polypeptides suchas anti-angiogenic polypeptides, neuroprotective polypeptides, and thelike, when discussed in the context of delivering a gene product to amammalian subject, and compositions therefor, refer to the respectiveintact polypeptide, or any fragment or genetically engineered derivativethereof, retaining the desired biochemical function of the intactprotein.

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids used in the disclosure methods areconventional and are as follows in Table 1.

TABLE 1 Amino acid abbreviations One-Letter Common Amino Acid SymbolAbbreviation Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acidD Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine G GlyHistidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine MMet Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include Thr (T),Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg(R).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydrogen ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain uncharged at physiological pH, but which has at least one bond inwhich the pair of electrons shared in common by two atoms is held moreclosely by one of the atoms. Genetically encoded polar amino acidsinclude Asn (N), Gln (Q), Ser (S) and Thr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Exemplary hydrophobic amino acids include Ile (I), Phe (F),Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), Pro (P),and proline analogues.

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as —OH,—SH, —CN, —F, —Cl, —Br, —I, —NO2, —NO, —NH2, —NHR, —NRR, —C(O)R,—C(O)OH, —C(O)OR, —C(O)NH2, —C(O)NHR, —C(O)NRR and the like where each Ris independently (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6)alkenyl, substituted (C1-C6) alkenyl, (C1-C6) alkynyl, substituted(C1-C6) alkynyl, (C1-C21)) aryl, substituted (C5-C20) aryl, (C6-C26)alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl,substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl orsubstituted 6-26 membered alkheteroaryl. Genetically encoded aromaticamino acids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain uncharged at physiological pH and which has bonds in which thepair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

“Aliphatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

The term “non-naturally” with regard to amino acids can include anyamino acid molecule not included as one of the 20 amino acids listed inTable 1 above as well as any modified or derivatized amino acid known toone of skill in the art. Non-naturally amino acids can include but arenot limited to β-alanine, α-amino butyric acid, γ-amino butyric acid,γ-(aminophenyl) butyric acid, α-amino isobutyric acid, ε-amino caproicacid, 7-amino heptanoic acid, β-aspartic acid, aminobenzoic acid,aminophenyl acetic acid, aminophenyl butyric acid, γ-glutamic acid,cysteine (ACM), ε-lysine, methionine sulfone, norleucine, norvaline,ornithine, d-ornithine, p-nitro-phenylalanine, hydroxy proline,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, and thioproline.

The term “variant” or “variants”, with regard to polypeptides, such ascapsid polypeptides refers to a polypeptide sequence differing by atleast one amino acid from a parent polypeptide sequence, also referredto as a non-variant polypeptide sequence (also referred to as anon-variant parent polypeptide). In some embodiments, the polypeptide isa capsid polypeptide and the variant differs by at least one amino acidsubstitution. Amino acids also include naturally occurring andnon-naturally occurring amino acids as well as derivatives thereof.Amino acids also include both D and L forms.

An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell,or other substance refers to a preparation of the substance devoid of atleast some of the other components present where the substance or asimilar substance naturally occurs or from which it is initiallyprepared. Thus, for example, an isolated substance may be prepared byusing a purification technique to enrich it from a source mixture.Enrichment can be measured on an absolute basis, such as weight pervolume of solution, or it can be measured in relation to a second,potentially interfering substance present in the source mixture.Increasing enrichments of the embodiments of this invention areincreasingly more isolated. An isolated plasmid, nucleic acid, vector,virus, host cell, or other substance is in some embodiments purified,e.g., from about 80% to about 90% pure, at least about 90% pure, atleast about 95% pure, at least about 98% pure, or at least about 99%, ormore, pure.

By the term “highly conserved”, it is meant at least about 80% identity,preferably at least about 90% identity, and more preferably, over about97% identity, is conserved. Identity is readily determined by one ofskill in the art by resort to algorithms and computer programs known bythose of skill in the art.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome,the full-length of a gene coding sequence, or a fragment of at leastabout 500 to 5000 nucleotides, is desired. However, identity amongsmaller fragments, e.g. of at least one or more nucleotides, may also bedesired. Similarly, “percent sequence identity” may be readilydetermined for amino acid sequences, over the full-length of a protein,or a fragment thereof. Suitably, a fragment is at least about 8 aminoacids in length, and may be up to the length of the protein is aminoacids.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subjectpredisposed to the disease or at risk of acquiring the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; and (c) relieving the disease, i.e., causingregression of the disease.

The terms “individual,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

The terms “pharmaceutically acceptable” and “physiologically acceptable”mean a biologically acceptable formulation, gaseous, liquid or solid, ormixture thereof, suitable for one or more routes of administration, invivo delivery or contact. A “pharmaceutically acceptable” or“physiologically acceptable” composition is a material that is notbiologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing substantial undesirablebiological effects. Thus, such a pharmaceutical composition may be used,for example in administering a variant AAV capsid, an AAV vector, or anAAV virion as disclosed herein, or cell transformed with an AAV, to asubject.

The phrase a “unit dosage form” as used herein refers to physicallydiscrete units suited as unitary dosages for the subject to be treated;each unit containing a predetermined quantity optionally in associationwith a pharmaceutical carrier (excipient, diluent, vehicle or fillingagent) which, when administered in one or more doses, produces a desiredeffect (e.g., prophylactic or therapeutic effect). In some embodiments,unit dosage forms may be within, for example, ampules and vials,including a liquid composition, or a composition in a freeze-dried orlyophilized state; a sterile liquid carrier, for example, can be addedprior to administration or delivery in vivo. Individual unit dosageforms can be included in multi-dose kits or containers. Variant AAVcapsids, AAV vectors, or AAV virions, and pharmaceutical compositionsthereof can be packaged in single or multiple unit dosage form for easeof administration and uniformity of dosage.

A “therapeutically effective amount” will fall in a relatively broadrange determinable through experimentation and/or clinical trials. Forexample, for in vivo injection, e.g., injection directly into the tissueor vaculature of a subject (for example, liver tissue or veins), atherapeutically effective dose will be on the order of from about 10⁶ toabout 10¹⁵ AAV virions, e.g., from about 10⁸ to 10¹² AAV virions. Othereffective dosages can be readily established by one of ordinary skill inthe art through routine trials establishing dose response curves.

An “effective amount” or “sufficient amount” refers to an amountproviding, in single or multiple doses, alone or in combination, withone or more other compositions (therapeutic agents such as a drug),treatments, protocols, or therapeutic regimens agents (including, forexample, vaccine regimens), a detectable response of any duration oftime (long or short term), an expected or desired outcome in or abenefit to a subject of any measurable or detectable degree or for anyduration of time (e.g., for minutes, hours, days, months, years, orcured).

The doses of an “effective amount” or “sufficient amount” for treatment(e.g., to ameliorate or to provide a therapeutic benefit or improvement)typically are effective to provide a response to one, multiple or alladverse symptoms, consequences or complications of the disease, one ormore adverse symptoms, disorders, illnesses, pathologies, orcomplications, for example, caused by or associated with the disease, toa measurable extent, although decreasing, reducing, inhibiting,suppressing, limiting or controlling progression or worsening of thedisease is also a satisfactory outcome.

“Prophylaxis” and grammatical variations thereof mean a method in whichcontact, administration or in vivo delivery to a subject is prior todisease. Administration or in vivo delivery to a subject can beperformed prior to development of an adverse symptom, condition,complication, etc. caused by or associated with the disease. Forexample, a screen (e.g., genetic) can be used to identify such subjectsas candidates for the described methods and uses, but the subject maynot manifest the disease. Such subjects therefore include those screenedpositive for an insufficient amount or a deficiency in a functional geneproduct (protein), or producing an aberrant, partially functional ornon-functional gene product (protein), leading to disease; and subjectsscreening positive for an aberrant, or defective (mutant) gene product(protein) leading to disease, even though such subjects do not manifestsymptoms of the disease.

The phrase “enhanced neutralization profile” refers to the ability of anAAV vector or virion to better evade neutralizing antibody binding inthe subject. In some instances, fewer neutralization antibodies allowfor the AAV infection to allow for higher levels of transduction, makingthe variant AAV capsid polypeptides, AAV vectors, and AAV virions of thepresent invention better suited for gene therapy purposes.

The phrases “tropism” and “transduction” are interrelated, but there aredifferences. The term “tropism” as used herein refers to the ability ofan AAV vector or virion to infect one or more specified cell types, butcan also encompass how the vector functions to transduce the cell in theone or more specified cell types; i.e., tropism refers to preferentialentry of the AAV vector or virion into certain cell or tissue type(s)and/or preferential interaction with the cell surface that facilitatesentry into certain cell or tissue types, optionally and preferablyfollowed by expression (e.g., transcription and, optionally,translation) of sequences carried by the AAV vector or virion in thecell, e.g., for a recombinant virus, expression of the heterologousnucleotide sequence(s). As used herein, the term “transduction” refersto the ability of an AAV vector or virion to infect one or moreparticular cell types; i.e., transduction refers to entry of the AAVvector or virion into the cell and the transfer of genetic materialcontained within the AAV vector or virion into the cell to obtainexpression from the vector genome. In some cases, but not all cases,transduction and tropism may correlate.

The term “tropism profile” refers to the pattern of transduction of oneor more target cells, tissues and/or organs. For example, some shuffledAAV capsids (variant AAV capsid polypeptides) provide for efficienttransduction of human liver tissue or hepatocyte cells (i.e., humanhepatocyte cells). Conversely, some shuffled AAV capsids have only lowlevel transduction of skeletal muscle (e.g., quadriceps muscle),diaphragm muscle and/or cardiac muscle tissue, gonads and/or germ cells.The variant AAV capsid polypeptides disclosed herein provide forefficient and/or enhanced transduction of human liver tissue orhepatocyte cells (i.e., human hepatocyte cells).

Unless indicated otherwise, “efficient transduction” or “efficienttropism,” or similar terms, can be determined by reference to a suitablecontrol (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%,110%, 125%, 150%, 175%, or 200% or more of the transduction or tropism,respectively, of the control). Suitable controls will depend on avariety of factors including the desired tropism profile. Similarly, itcan be determined if a capsid and/or virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an AAV virion” includes aplurality of such virions and reference to “a host cell” includesreference to one or more host cells and equivalents thereof known tothose skilled in the art, and so forth. It is further noted that theclaims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

Before the invention is further described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

AAV Capsid and Vector Features

AAV vectors of the present invention have numerous features. In someembodiments, the vectors comprise nucleic acid sequences encoding forvariant capsid polypeptides. Such AAV vectors and their features aredescribed in detail below.

An exemplary AAV vector of the present invention comprises a nucleicacid encoding for a variant AAV capsid protein differing in amino acidsequence by at least one amino acid from a wild-type or non-variantparent capsid protein. The amino acid difference(s) can be located in asolvent accessible site in the capsid, e.g., a solvent-accessible loop,or in the lumen (i.e., the interior space of the AAV capsid). In someembodiments, the lumen includes the interior space of the AAV capsid.For example, the amino acid substitution(s) can be located in a GH loopin the AAV capsid polypeptide. In some embodiments, the variant capsidpolypeptide comprises an amino acid substitution in AAV1, AAV2, AAV3,AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsid polypeptides.

In some embodiments, the present invention provides an isolated nucleicacid comprising a nucleotide sequence that encodes a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85% at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, to non-variant capsid amino acid sequences or tosub-portions of a non-variant parent capsid polypeptide sequence, andexhibits an enhanced neutralization profile as compared to a vectorencoding a non-variant parent capsid polypeptide. In some embodiments,the present invention provides an isolated nucleic acid comprising anucleotide sequence that encodes a variant adeno-associated virus (AAV)capsid protein, where the variant AAV capsid protein comprises an aminoacid sequence having at least about 85% at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, to parentalnon-variant capsid amino acid sequences or to sub-portions of anon-variant parent capsid polypeptide sequence, such as wild-type AAV1,AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsidpolypeptides, and where the variant capsid polypeptide exhibits anenhanced neutralization profile as compared to a vector encoding anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide comprises one or more regions or sub-portions fromnon-variant parent capsid polypeptide sequences from AAV serotypes 1, 2,3b, and 6 (i.e., AAV1, AAV2, AAV3b, and AAV6). In some embodiments, thepresent invention provides a variant capsid polypeptide according to theabove.

In some embodiments, the present invention provides an isolated nucleicacid comprising a nucleotide sequence that encodes a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, to non-variant capsid amino acid sequences orto sub-portions of a non-variant parent capsid polypeptide sequence, andexhibits increased transduction or tropism in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells) as compared to a vectorencoding a non-variant parent capsid polypeptide. In some embodiments,the present invention provides an isolated nucleic acid comprising anucleotide sequence that encodes a variant adeno-associated virus (AAV)capsid protein, where the variant AAV capsid protein comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, to parentalnon-variant capsid amino acid sequences or to sub-portions of anon-variant parent capsid polypeptide sequence, such as wild-type AAV1,AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsidpolypeptides, and where the variant capsid polypeptide exhibitsincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a vector encoding anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide comprises one or more regions or sub-portions fromnon-variant parent capsid polypeptide sequences from AAV serotypes 1, 2,3b, and 6 (i.e., AAV1, AAV2, AAV3b, and AAV6). In some embodiments, thepresent invention provides a variant capsid polypeptide according to theabove. In some embodiments, the parental serotypes that contributed themost to the variant capsid polypeptides included AAV2, 3b, 1 and 6, inthat order. In some embodiments, the variant capsid polypeptide does notcomprise one or more regions or sub-portions from AAV4, 5, 8, 9_hu14,bovine or avian. In some embodiments, the variant capsid polypeptidedoes not comprise one or more regions or sub-portions from AAV8.

In some embodiments, the present invention provides an isolated nucleicacid comprising a nucleotide sequence that encodes a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, to non-variant capsid amino acid sequences orto sub-portions of a non-variant parent capsid polypeptide sequence, andexhibits both an enhanced neutralization profile and increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) as compared to a vector encoding a non-variantparent capsid polypeptide. In some embodiments, the present inventionprovides an isolated nucleic acid comprising a nucleotide sequence thatencodes a variant adeno-associated virus (AAV) capsid protein, where thevariant AAV capsid protein comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, to parental non-variant capsid aminoacid sequences or to sub-portions of a non-variant parent capsidpolypeptide sequence, such as wild-type AAV1, AAV2, AAV3, AAV3b, AAV4,AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsid polypeptides, and where thevariant capsid polypeptide exhibits both an enhanced neutralizationprofile and increased transduction or tropism in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells) as compared to anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide comprises one or more regions or sub-portions fromnon-variant parent capsid polypeptide sequences from AAV serotypes 1, 2,3b, and 6 (i.e., AAV1, AAV2, AAV3b, and AAV6). In some embodiments, theparental serotypes that contributed the most to the variant capsidpolypeptides included AAV2, 3b, 1 and 6, in that order. In someembodiments, the variant capsid polypeptide does not comprise one ormore regions or sub-portions from AAV4, 5, 8, 9_hu14, bovine or avian.In some embodiments, the variant capsid polypeptide does not compriseone or more regions or sub-portions from AAV8. In some embodiments, thepresent invention provides a variant capsid polypeptide according to theabove.

In some embodiments, a subject AAV vector can encode a variant capsidpolypeptide having an amino acid sequence of at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99%, or 100%, amino acid sequence identity to non-variant parentcapsid polypeptide or to sub-portions of a non-variant parent capsidpolypeptide. In some embodiments, the variant capsid polypeptide isencoded by other vectors/plasmids known in the art. In some embodiments,the present invention provides a variant capsid polypeptide according tothe above.

In some embodiments, the present invention provides a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, to non-variant capsid amino acid sequences orto sub-portions of a non-variant parent capsid polypeptide sequence, andexhibits an enhanced neutralization profile as compared a non-variantparent capsid polypeptide. In some embodiments, the present inventionprovides a variant adeno-associated virus (AAV) capsid protein, wherethe variant AAV capsid protein comprises an amino acid sequence havingat least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, to parental non-variant capsidamino acid sequences or to sub-portions of a non-variant parent capsidpolypeptide sequence, such as wild-type AAV1, AAV2, AAV3, AAV3b, AAV4,AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsid polypeptides, and where thevariant capsid polypeptide exhibits an enhanced neutralization profileas compared to a non-variant parent capsid polypeptide. In someembodiments, the variant capsid polypeptide comprises one or moreregions or sub-portions from non-variant parent capsid polypeptidesequences from AAV serotypes 1, 2, 3b, and 6 (i.e., AAV1, AAV2, AAV3b,and AAV6).

In some embodiments, the present invention provides a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, to non-variant capsid amino acid sequences orto sub-portions of a non-variant parent capsid polypeptide sequence, andexhibits increased transduction or tropism in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells) as compared tonon-variant parent capsid polypeptide. In some embodiments, the presentinvention provides a variant adeno-associated virus (AAV) capsidprotein, where the variant AAV capsid protein comprises an amino acidsequence having at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, to parentalnon-variant capsid amino acid sequences or to sub-portions of anon-variant parent capsid polypeptide sequence, such as wild-type AAV1,AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsidpolypeptides, and where the variant capsid polypeptide exhibitsincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a non-variant parentcapsid polypeptide. In some embodiments, the variant capsid polypeptidecomprises one or more regions or sub-portions from non-variant parentcapsid polypeptide sequences from AAV serotypes 1, 2, 3b, and 6 (i.e.,AAV1, AAV2, AAV3b, and AAV6). In some embodiments, the parentalserotypes that contributed the most to the variant capsid polypeptidesincluded AAV2, 3b, 1 and 6, in that order. In some embodiments, thevariant capsid polypeptide does not comprise one or more regions orsub-portions from AAV4, 5, 8, 9_hu14, bovine or avian. In someembodiments, the variant capsid polypeptide does not comprise one ormore regions or sub-portions from AAV8.

In some embodiments, the present invention provides a variantadeno-associated virus (AAV) capsid protein, where the variant AAVcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, to non-variant capsid amino acid sequences orto sub-portions of a non-variant parent capsid polypeptide sequence, andexhibits both an enhanced neutralization profile and increasedtransduction or tropism in human liver tissue or hepatocyte cells (i.e.,human hepatocyte cells) as compared to a non-variant parent capsidpolypeptide. In some embodiments, the present invention provides avariant adeno-associated virus (AAV) capsid protein, where the variantAAV capsid protein comprises an amino acid sequence having at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, to parental non-variant capsid amino acidsequences or to sub-portions of a non-variant parent capsid polypeptidesequence, such as wild-type AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6,AAV7, AAV8, or AAV9_hu14 capsid polypeptides, and where the variantcapsid polypeptide exhibits both an enhanced neutralization profile andincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a non-variant parentcapsid polypeptide. In some embodiments, the variant capsid polypeptidecomprises one or more regions or sub-portions from non-variant parentcapsid polypeptide sequences from AAV serotypes 1, 2, 3b, and 6 (i.e.,AAV1, AAV2, AAV3b, and AAV6). In some embodiments, the parentalserotypes that contributed the most to the variant capsid polypeptidesincluded AAV2, 3b, 1 and 6, in that order. In some embodiments, thevariant capsid polypeptide does not comprise one or more regions orsub-portions from AAV4, 5, 8, 9_hu14, bovine or avian. In someembodiments, the variant capsid polypeptide does not comprise one ormore regions or sub-portions from AAV8.

In some embodiments, the variant capsid polypeptides exhibit substantialhomology or “substantial similarity,” when referring to amino acids orfragments thereof, indicates that, when optimally aligned withappropriate amino acid insertions or deletions with another amino acid(or its complementary strand), there is amino acid sequence identity inat least about 95% to about 99% of the aligned sequences. In someembodiments, the homology is over full-length sequence, or a polypeptidethereof, e.g., a capsid protein, or a fragment thereof of at least 8amino acids, or more desirably, at least about 15 amino acids in length,including sub-portions of a non-variant parent capsid polypeptidesequence. For example, the variant capsid polypeptide can comprise anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a non-variant parent capsid polypeptidesequence or to sub-portions of a non-variant parent capsid polypeptide.In some embodiments, the variant AAV capsid protein comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, to parentalnon-variant capsid amino acid sequences or to sub-portions of anon-variant parent capsid polypeptide sequence, such as wild-type AAV1,AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsidpolypeptides, and where the variant capsid polypeptide exhibitsincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a vector encoding anon-variant parent capsid polypeptide. In some embodiments, the variantAAV capsid protein comprises an amino acid sequence having at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, to parental non-variant capsid amino acidsequences or to sub-portions of a non-variant parent capsid polypeptidesequence, such as wild-type AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6,AAV7, AAV8, or AAV9_hu14 capsid polypeptides, and where the variantcapsid polypeptide exhibits an enhanced neutralization profile ascompared to a vector encoding a non-variant parent capsid polypeptide.In some embodiments, the variant AAV capsid protein comprises an aminoacid sequence having at least about 85% at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, to parentalnon-variant capsid amino acid sequences or to sub-portions of anon-variant parent capsid polypeptide sequence, such as wild-type AAV1,AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9_hu14 capsidpolypeptides, and where the variant capsid polypeptide exhibits both anenhanced neutralization profile as compared to a vector encoding anon-variant parent capsid polypeptide and increased transduction ortropism in human liver tissue or hepatocyte cells (i.e., humanhepatocyte cells) as compared to a vector encoding a non-variant parentcapsid polypeptide. In some embodiments, the variant capsid polypeptidecomprises one or more regions or sub-portions from non-variant parentcapsid polypeptide sequences from AAV serotypes 1, 2, 3b, and 6 (i.e.,AAV1, AAV2, AAV3b, and AAV6). In some embodiments, the parentalserotypes that contributed the most to the variant capsid polypeptidesincluded AAV2, 3b, 1 and 6, in that order. In some embodiments, thevariant capsid polypeptide does not comprise one or more regions orsub-portions from AAV4, 5, 8, 9_hu14, bovine or avian. In someembodiments, the variant capsid polypeptide does not comprise one ormore regions or sub-portions from AAV8. In some embodiments the variantcapsid polypeptide sequence comprises SEQ ID NOs:2, 4, 6, or 8, asprovided in Table 2 below. In some embodiments, the variant capsidpolypeptide sequence is encoded by SEQ ID NOs:1, 3, 5, or 7, as providedin Table 2 below. In some embodiments, the variant AAV capsidpolypeptide is selected from the group consisting of AAV-NP84 (SEQ IDNO:2), AAV-NP59 (SEQ ID NO:4), and AAV-NP40 (SEQ ID NO:6). In someembodiments, the variant AAV capsid polypeptide is selected from thegroup consisting of AAV-NP59 (SEQ ID NO:4), and AAV-NP40 (SEQ ID NO:6).In some embodiments, the variant AAV capsid polypeptide is AAV-NP59 (SEQID NO:4). In some embodiments, the variant AAV capsid polypeptide isAAV-NP40 (SEQ ID NO:6).

TABLE 2 Variant Capsid Sequences Description SEQ ID NO: SequenceNucleic acid >AAV-NP84-nt sequence forATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAAV-NP84AGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTSEQ IDTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCNO: 1GCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCAGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCCACCTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAGAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGGGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA Amino acid >AAV-NP84-aasequence forMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAAV-NP84AALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPSEQ IDGKKRPVEHSPVEPDSSSGTGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGTNTMATGSGAPNO: 2MADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEEFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQGGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Nucleic acid >AAV-NP59-ntsequence forATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAAV-NP59AGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTSEQ IDTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCNO: 3GCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCGACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGACCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA Amino acid >AAV-NP59-aasequence forMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAAV-NP59AALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPSEQ IDGKKRPVEHSPVEPDSSSGTGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGTNTMATGSGAPNO: 4MADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVDTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Nucleic acid >AAV-NP40-ntsequence forATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATAAGACAGTGGTGGAAAV-NP40AGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTSEQ IDTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCNO: 5GCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCCACCTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAGAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA Amino acid >AAV-NP40-aasequence forMAADGYLPDWLEDNLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAAV-NP40AALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPSEQ IDGKKRPVEHSPVEPDSSSGTGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGTNTMATGSGAPNO: 6MADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEEFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Nucleic acid >AAV-NP30-ntsequence forATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAAV-NP30AGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTSEQ IDTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCNO: 7GCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCAGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCCACCTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTTAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAGAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA Amino acid >AAV-NP30-aasequence forMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAAV-NP30AALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPSEQ IDGKKRPVEHSPVEPDSSSGTGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGTNTMATGSGAPNO: 8MADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEEFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Nucleic acid >AAV-LK03-ntsequence forATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTTTCTGAAGGCATTCGAGAGTGGTGGGLK03CGCTGCAACCTGGAGCCCCTAAACCCAAGGCAAATCAACAACATCAGGACAACGCTCGGGGTCTTGTGCTSEQ IDTCCGGGTTACAAATACCTCGGACCCGGCAACGGACTCGACAAGGGGGAACCCGTCAACGCAGCGGACGCGNO: 9GCAGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGTGACAACCCCTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGATCAGTCTCCTCAGGAACCGGACTCATCATCTGGTGTTGGCAAATCGGGCAAACAGCCTGCCAGAAAAAGACTAAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCACAAGTTTGGGATCTAATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCAGGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACGGACTCGGAGTATCAGCTCCCGTACGTGCTCGGGTCGGCGCACCAAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGGTCCCTCAGTATGGATACCTCACCCTGAACAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTACTTCCCTTCGCAGATGCTAAGGACTGGAAATAACTTCCAATTCAGCTATACCTTCGAGGATGTACCTTTTCACAGCAGCTACGCTCACAGCCAGAGTTTGGATCGCTTGATGAATCCTCTTATTGATCAGTATCTGTACTACCTGAACAGAACGCAAGGAACAACCTCTGGAACAACCAACCAATCACGGCTGCTTTTTAGCCAGGCTGGGCCTCAGTCTATGTCTTTGCAGGCCAGAAATTGGCTACCTGGGCCCTGCTACCGGCAACAGAGACTTTCAAAGACTGCTAACGACAACAACAACAGTAACTTTCCTTGGACAGCGGCCAGCAAATATCATCTCAATGGCCGCGACTCGCTGGTGAATCCAGGACCAGCTATGGCCAGTCACAAGGACGATGAAGAAAAATTTTTCCCTATGCACGGCAATCTAATATTTGGCAAAGAAGGGACAACGGCAAGTAACGCAGAATTAGATAATGTAATGATTACGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAGCAGTATGGAACTGTGGCAAATAACTTGCAGAGCTCAAATACAGCTCCCACGACTAGAACTGTCAATGATCAGGGGGCCTTACCTGGCATGGTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCAAAGATTCCTCACACGGATGGACACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATCCGCCTCCTCAAATCATGATCAAAAATACTCCGGTACCGGCAAATCCTCCGACGACTTTCAGCCCGGCCAAGTTTGCTTCATTTATCACTCAGTACTCCACTGGACAGGTCAGCGTGGAAATTGAGTGGGAGCTACAGAAAGAAAACAGCAAACGTTGGAATCCAGAGATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTAGACACTAATGGTGTTTATAGTGAACCTCGCCCCATTGGCACCCGTTACCTTACCCGTCCCCTGTAA Amino acid >AAV-LK03-aasequence forMAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADALK03AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPSEQ IDGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPNO: 10MADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL* Nucleic acid >AAV-DJ-ntsequence forATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCtCTGAAGGAATAAGACAGTGGTGGADJAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTSEQ IDTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCNO: 11GCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTtGGTCTGGTTGAGGAAGCGGCTAAGACGGcTCcTGGAAaGAaGAGgCcTGTAGAGCACTCTCCTGTGGAGCCAGACTCcTCcTCGGgAACCGGAAAgGCGGgCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAaTAaCcTCACcAGCACcATCcAGgTGTTtACGgACTCgGAGTACCAGCTGCCGTACGTtCTCGGCTCTGcCCACCAGGGCTGcCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGgaCCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACTCAAaCAaCAGgAGgCACGACaAATACGCAGACTCTGGGCTTCAGCCAaGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA Amino acid >AAV-DJ-aasequence forMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADADJAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPSEQ IDGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPNO: 12MADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL*

In some embodiments, the variant AAV capsid polypeptides of theinvention exhibit increased transduction in human liver tissue orhepatocyte cells (i.e., human hepatocyte cells) as compared to anon-variant parent capsid polypeptide. In some embodiments, transductionis increased by about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, 65%, about 70%%, about 75%, about 80%, about 85%, about 90%, about95%, about 99%, or about 100%. In some embodiments, transduction isincreased by about 5% to about 80%, about 10% to about 70%, about 20% toabout 60%, about 30% to about 60%, or about 40% to about 50%.

In some embodiments, the variant AAV capsid polypeptides of theinvention exhibit increased tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a non-variant parentcapsid. In some embodiments, tropism is increased by about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. Insome embodiments, tropism is increased by about 5% to about 80%, about10% to about 70%, about 20% to about 60%, about 30% to about 60%, orabout 40% to about 50%.

In some embodiments, the variant AAV capsid polypeptides of theinvention exhibit an enhanced neutralization profile as compared to anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide of the invention further exhibits an enhancedneutralization profile against pooled human immunoglobulins as comparedto a non-variant parent capsid polypeptide. In some embodiments, theneutralization profile is enhanced by about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, 65%, about 70%%, about 75%, about 80%, about85%, about 90%, about 95%, about 99%, or about 100%. In someembodiments, the neutralization profile is enhanced by about 5% to about80%, about 10% to about 70%, about 20% to about 60%, about 30% to about60%, or about 40% to about 50%. In some embodiments, an enhancedneutralization profile is determined by a reduction in the generation ofneutralizing antibodies in a host. In some embodiments, the reduction ingeneration of neutralizing antibodies is a reduction of about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. Insome embodiments, the reduction in generation of neutralizing antibodiesis a reduction of about 5% to about 80%, about 10% to about 70%, about20% to about 60%, about 30% to about 60%, or about 40% to about 50%.

In some embodiments, the enhanced neutralization profiles are againstpooled human immunoglobulins (i.e., IgG or IVIG, commercially availableas Gammagard IVIG). In some embodiments, the ratio of variant AAV capsidpolypeptides bound to IVIG is determined as an indicator of an enhancedneutralization profile and methods for determining neutralizationprofiles are known in the art (see, for example, Grimm, et al., J.Virol. 82(12): 5887-5911 (2008); Arbetman, et al. J. Virol.79(24):15238-15245 (2005)). In some embodiments, a low ratio of variantAAV capsid polypeptides bound to IVIG as compared to variant AAV capsidpolypeptides unbound to IVIG is indicative of an enhanced neutralizationprofile (see, for example, FIG. 6). In some embodiments, the enhancedneutralization profile is determined by determining the concentration ofIVIG needed to decrease the signal generated from a cell transduced by avariant AAV capsid by about 50% as compared to a control sample signal.In some embodiments, the control sample signal is the signal generatedfrom a cell transduced by the variant AAV capsid in the absence of IVIG.In some embodiments, the concentration is a concentration of IVIGsufficient to decrease the signal generated from a cell transduced by avariant AAV capsid by at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, or at least about 95% as compared to thecontrol sample signal. In some embodiments, the concentration is aconcentration of IVIG sufficient to decrease the signal by at leastabout 50% as compared to the control sample signal. In some embodiments,the concentration is a concentration of IVIG sufficient to decrease thesignal generated from a cell transduced by a variant AAV capsid by atleast about 40% to at least about 95% as compared to the control samplesignal. In some embodiments, the concentration is a concentration ofIVIG sufficient to decrease the signal generated from a cell transducedby a variant AAV capsid by at least about 50% to at least about 90% ascompared to the control sample signal. In some embodiments, theconcentration is a concentration of IVIG sufficient to decrease thesignal generated from a cell transduced by a variant AAV capsid by atleast about 50% to at least about 85% as compared to the control samplesignal. In some embodiments, the concentration is a concentration ofIVIG sufficient to decrease the signal generated from a cell transducedby a variant AAV capsid by at least about 50% to at least about 80% ascompared to the control sample signal. In some embodiments, theconcentration is a concentration of IVIG sufficient to decrease thesignal generated from a cell transduced by a variant AAV capsid by atleast about 55% to at least about 75% as compared to the control samplesignal. In some embodiments, the concentration is a concentration ofIVIG sufficient to decrease the signal generated from a cell transducedby a variant AAV capsid by at least about 50% to at least about 70% ascompared to the control sample signal. In some embodiments, theconcentration is a concentration of IVIG sufficient to decrease thesignal generated from a cell transduced by a variant AAV capsid by atleast about 50% to at least about 65% as compared to the control samplesignal. In some embodiments, the concentration is a concentration ofIVIG sufficient to decrease the signal generated from a cell transducedby a variant AAV capsid by at least about 50% to at least about 60% ascompared to the control sample signal. In some embodiments, theconcentration is a concentration of IVIG sufficient to decrease thesignal generated from a cell transduced by a variant AAV capsid by atleast about 50% to at least about 55% as compared to the control samplesignal. In some embodiments, the signal generated is any detectablesignal. In some embodiments, the signal generated is a fluorescentsignal. In some embodiments, the signal generated is from GFP (greenfluorescent protein), CFP (cyan fluorescent protein), YFP (yellowfluorescent protein), and RFP (red fluorescent protein). In someembodiments, the signal generated is a bioluminescent signal. In someembodiments, the signal generated is from luciferase (Firefly luciferaseor Renilla luciferase).

In some embodiments, the variant AAV capsid polypeptides of theinvention further exhibit increased transduction or tropism in one ormore human stem cell types as compared to a non-variant parent capsidpolypeptide. In some embodiments, the human stem cell types include butare not limited to embryonic stem cells, adult tissue stem cells (i.e.,somatic stem cells), bone marrow, progenitor cells, induced pluripotentstem cells, and reprogrammed stem cells. In some embodiments, adult stemcells can include organoid stem cells (i.e., stem cells derived from anyorgan or organ system of interest within the body). Organs of the bodyinclude for example but are not limited to skin, hair, nails, sensereceptors, sweat gland, oil glands, bones, muscles, brain, spinal cord,nerve, pituitary gland, pineal gland, hypothalamus, thyroid gland,parathyroid, thymus, adrenals, pancreas (islet tissue), heart, bloodvessels, lymph nodes, lymph vessels, thymus, spleen, tonsils, nose,pharynx, larynx, trachea, bronchi, lungs, mouth, pharynx, esophagus,stomach, small intestine, large intestine, rectum, anal canal, teeth,salivary glands, tongue, liver, gallbladder, pancreas, appendix,kidneys, ureters, urinary bladder, urethra, testes, ductus (vas)deferens, urethra, prostate, penis, scrotum, ovaries, uterus, uterine(fallopian) tubes, vagina, vulva, and mammary glands (breasts). Organsystems of the body include but are not limited to the integumentarysystem, skeletal system, muscular system, nervous system, endocrinesystem, cardiovascular system, lymphatic system, respiratory system,digestive system, urinary system, and reproductive system. In someembodiments, transduction and/or tropism is increased by about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. Insome embodiments, transduction and/or tropism is increased by about 5%to about 80%, about 10% to about 70%, about 20% to about 60% or about30% to about 60%.

In some embodiments, the variant AAV capsid polypeptides of theinvention further exhibit increased transduction or tropism in one ormore non-liver human tissues as compared to a non-variant parent capsidpolypeptide. In some embodiments, the variant capsid polypeptide furtherexhibits increased transduction in one or more non-liver human tissuesas compared to a vector encoding a non-variant parent capsidpolypeptide. In some embodiments, the variant capsid polypeptide furtherexhibits increased tropism in one or more non-liver human tissues ascompared to a vector encoding a non-variant parent capsid polypeptide.In some embodiments, transduction and/or tropism is increased by about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, 65%, about 70%%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, orabout 100%. In some embodiments, transduction and/or tropism isincreased by about 5% to about 80%, about 10% to about 70%, about 20% toabout 60% or about 30% to about 60%.

In some embodiments, the variant AAV capsid polypeptides of theinvention further exhibit increased transduction or tropism in one ormore non-liver human tissues as compared to a non-variant parent capsidpolypeptide. In some embodiments, the variant capsid polypeptide furtherexhibits increased transduction in one or more non-liver human tissuesas compared to a vector encoding a non-variant parent capsidpolypeptide. In some embodiments, the variant capsid polypeptide furtherexhibits increased tropism in one or more non-liver human tissues ascompared to a vector encoding a non-variant parent capsid polypeptide.In some embodiments, transduction and/or tropism is increased by about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, 65%, about 70%%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, orabout 100%. In some embodiments, transduction and/or tropism isincreased by about 5% to about 80%, about 10% to about 70%, about 20% toabout 60% or about 30% to about 60%.

In some embodiments, the variant capsid polypeptide contains a VP1comprising AAV1, AAV3b, AAV6, AAV8 or AAV9_hu14. In some embodiments,the variant capsid polypeptide contains a VP2 comprising AAV2. In someembodiments, the variant capsid polypeptide contains a unique region ofVP2 from AAV2. In some embodiments, the variant capsid polypeptidecontains a VP3 comprising AAV1, AAV2, AAV3b and AAV6. In someembodiments, the variant capsid polypeptide contains a VP1 comprisingAAV2. In some embodiments, the variant capsid polypeptide contains a VP2comprising AAV2. In some embodiments, the variant capsid polypeptidecontains a VP3 comprising AAV1, AAV2, AAV3b and AAV6. In someembodiments, the variant capsid polypeptide is NP-40 (SEQ ID NO:6). Insome embodiments, the variant capsid polypeptide is NP-59 (SEQ ID NO:4).In some embodiments, there is a conserved contribution from AAV3b atpositions 326-426

(SEQ ID NO: 13) (NDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSY AH).

In some embodiments, the variant capsid polypeptide comprises the aminoacid sequence of 326-426 from AAV3b. In some embodiments, the variantcapsid polypeptide comprises the amino acid sequence of 326-426 fromAAV3b and exhibits enhanced human hepatic transduction. In someembodiments, the variant capsid polypeptide comprises the cylinder (fromAAV2). In some embodiments, the variant capsid polypeptide comprisescanyon (from AAV3b). In some embodiments, the variant capsid polypeptidecomprises one or more substitutions selected from the group consistingof K555E, N622D, and R611G. In some embodiments, the variant capsidpolypeptide comprises K555E. In some embodiments, the variant capsidpolypeptide comprises N622D. In some embodiments, the variant capsidpolypeptide comprises R611G. In some embodiments, the variant capsidpolypeptide comprises K555E and R611G. In some embodiments, the variantcapsid polypeptide comprises any combination of one or more of the abovefeatures.

As an exemplary embodiment of the variant capsid polypeptides of theinvention, NP40 is the most shuffled of the three, with the uniqueregion of VP1 from AAV1/3b/6/8/9, the unique region of VP2 derived fromAAV2, and finally VP3 with contributions from AAV2 and 3b as well as onede novo mutation (K555E). NP40 exhibits a conserved contribution fromAAV3b at positions 326-426. In some embodiments, this is the minimalstructural region from AAV3b for enhanced human hepatic transduction. Asanother exemplary embodiment of the variant capsid polypeptides of theinvention, NP59 is similar to NP40 but lacks the diverse VP1contributions and is instead composed of AAV2 in that sequence stretch.NP59 has the same VP2 and VP3 contributions as NP40 except for one denovo mutation (N622D). As another exemplary embodiment of the variantcapsid polypeptides of the invention, NP84 shares the unique regions ofVP1 and VP2 with NP59, but has a much larger contribution from AAV3b andless from AAV2 in VP3, as well as two de novo mutations (K555E andR611G).

In some embodiments, the variant capsid polypeptide sequence is selectedfrom the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ IDNO:4), AAV-NP40 (SEQ ID NO:6) and AAV-NP30 (SEQ ID NO:8). For NP84, theunique region of VP1 is composed of AAV2, the unique region of VP2 iscomposed of AAV2, and VP3 is composed of AAV1, AAV2, AAV3b, AAV6 andseveral de novo mutations. For NP59, the unique region of VP1 iscomposed of AAV2, the unique region of VP2 is composed of AAV2, and VP3is composed of AAV1, AAV2, AAV3b, AAV6 and several de novo mutations.For NP40, the unique region of VP1 is composed of AAV1, AAV3b, AAV6,AAV8 and/or AAV9 at equal probability, the unique region of VP2 iscomposed of AAV2, and VP3 is composed of AAV1, AAV2, AAV3b, AAV6 andseveral de novo mutations. For NP30, the unique region of VP1 iscomposed of AAV2, the unique region of VP2 is composed of AAV2, and VP3is composed of AAV1, AAV2, AAV3b, AAV6 and several de novo mutations. Insome embodiments, the variant capsid polypeptide comprises one or moresubstitutions (i.e., de novo mutations) selected from the groupconsisting of K555E, N622D, and R611G. In some embodiments, the variantcapsid polypeptide comprises K555E. In some embodiments, the variantcapsid polypeptide comprises N622D. In some embodiments, the variantcapsid polypeptide comprises R611G. In some embodiments, the variantcapsid polypeptide comprises K555E and R611G. In some embodiments, thevariant capsid polypeptide comprises any combination of one or more ofthe above features and/or substitutions (i.e., de novo mutations).

The present invention also provides for generating variant capsidpolypeptides, such as AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ ID NO:4),AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8). These methods employknown techniques of library generation; however, the methods are novelin that they employ replication competent AAV vectors during the variantcapsid polypeptide generation (i.e., selection and evolution of thevariant capsid polypeptides). The present invention provides methods forgenerating variant AAV capsid polypeptides, wherein the variant capsidpolypeptides exhibits both an enhanced neutralization profile andincreased transduction or tropism in human liver tissue or hepatocytecells (i.e., human hepatocyte cells) as compared to a non-variant parentcapsid polypeptide, said method comprising:

-   -   a) generating a library of variant AAV capsid polypeptides,        wherein said variant AAV capsid polypeptides include a plurality        of variant AAV capsid polypeptide sequences from more than one        non-variant parent capsid polypeptide;    -   b) generating an AAV vector library by cloning said variant AAV        capsid polypeptide library into AAV vectors, wherein said AAV        vectors are replication competent AAV vectors;    -   c) screening said AAV vector library from b) for variant AAV        capsid polypeptides for both an enhanced neutralization profile        and increased transduction or tropism in human liver tissue or        hepatocyte cells (i.e., human hepatocyte cells) as compared to a        non-variant parent capsid polypeptide; and    -   d) selecting said variant AAV capsid polypeptides from c).

In some embodiments, the method further comprises e) determining thesequence of said variant capsid polypeptides from d).

In some embodiments, the variant AAV capsid polypeptides generated byscreening methods of the invention exhibit increased transduction inhuman liver tissue as compared to a non-variant parent capsidpolypeptide. In some embodiments, transduction is increased by about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about100%. In some embodiments, transduction is increased by about 5% toabout 80%, about 10% to about 70%, about 20% to about 60%, about 30% toabout 60%, or about 40% to about 50%.

In some embodiments, the variant AAV capsid polypeptide generated byscreening methods of the invention exhibits increased tropism in humanliver tissue as compared to a non-variant parent capsid. In someembodiments, tropism is increased by about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, 65%, about 70%%, about 75%, about 80%, about85%, about 90%, about 95%, about 99%, or about 100%. In someembodiments, tropism is increased by about 5% to about 80%, about 10% toabout 70%, about 20% to about 60%, about 30% to about 60%, or about 40%to about 50%.

In some embodiments, the variant AAV capsid polypeptides generated byscreening methods of the invention exhibit an enhanced neutralizationprofile as compared to a non-variant parent capsid polypeptide. In someembodiments, the neutralization profile is enhanced by about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about 75%,about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. Insome embodiments, the neutralization profile is enhanced by about 5% toabout 80%, about 10% to about 70%, about 20% to about 60%, about 30% toabout 60%, or about 40% to about 50%. In some embodiments, an enhancedneutralization profile is determined by reduced preexisting neutralizingantibodies in a host which cross-react with the new AAV capsidembodiment. In some embodiments, the reduction in preexistingneutralizing antibody cross-reactivity is about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, 65%, about 70%%, about 75%, about 80%,about 85%, about 90%, about 95%, about 99%, or about 100%. In someembodiments, the reduction in generation of neutralizing antibodies is areduction of about 5% to about 80%, about 10% to about 70%, about 20% toabout 60%, about 30% to about 60%, or about 40% to about 50%.

In some embodiments, the variant AAV capsid polypeptide generated byscreening methods of the invention further exhibits increasedtransduction or tropism in one or more non-liver human tissues ascompared to a non-variant parent capsid polypeptide. In someembodiments, the variant capsid polypeptide further exhibits increasedtransduction in one or more non-liver human tissues as compared to anon-variant parent capsid polypeptide. In some embodiments, the variantcapsid polypeptide further exhibits increased tropism in one or morenon-liver human tissues as compared to a non-variant parent capsidpolypeptide. In some embodiments, transduction and/or tropism isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,65%, about 70%%, about 75%, about 80%, about 85%, about 90%, about 95%,about 99%, or about 100%. In some embodiments, transduction and/ortropism is increased by about 5% to about 80%, about 10% to about 70%,about 20% to about 60% or about 30% to about 60%.

Transduction can be measured by techniques known in the art, including,for example, immunohistochemical analysis, including those described inExample 1 below, as well as other methods known in the art. In vitrotransduction analysis can be performed in human liver tissue cells orhepatocyte cells, including for example by measuring GFP expression (oranother marker gene) in order to determine transduction. In vivo or exvivo transduction analysis can be measured by techniques known in theart, including, for example, Firefly luciferase-based assays, includingfor example by measuring luciferase expression (or another marker gene)in order to determine transduction. In some embodiments, markerexpression from an AAV vector packaged with the variant capsidpolypeptides is compared to marker expression from an AAV vectorpackaged with the non-variant parent capsid polypeptides in order tocompare transduction efficiencies. In some embodiments, the transductionis compared for different cell types in order to determine tropism,i.e., compare transduction from an AAV vector packaged with the variantcapsid polypeptide to transduction from an AAV packaged with thenon-variant capsid polypeptide in at least two different cell types inorder to determine tropism for a particular cell type, sometimesreferred to as a tropism profile. In some embodiments, at least one celltype is human liver tissue cells or human hepatocyte cells. In someembodiments, at least one cell type is human liver tissue cells. In someembodiments, at least one cell type is human hepatocyte cells.

Such methods for generating the variant capsid polypeptides include DNAshuffling of capsid proteins, which begins with families of capsid genesfrom an array or plurality of AAV pseudo-species (for example, AAV1,AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 9_hu14, bovine AAV,avian AAV), that are enzymatically shuffled to create a diverse libraryof capsid genes that can be cloned back into an AAV shuttle plasmid andutilized to produce live replicating viral libraries (see, for example,FIG. 2). To maximize the likelihood that a shuffled capsid (i.e.,variant capsid polypeptides) could functionally transduce human livertissue or hepatocyte cells (i.e., human hepatocyte cells)—as compared toliver tissue or hepatocyte cells of model organisms typically used forpre-clinical evaluation—the invention contemplates performing a screenin humanized liver mice (see, for example, FIG. 3). To maximize thelikelihood that shuffled capsids (i.e., variant capsid polypeptides)exhibit enhanced neutralization profiles, the invention contemplatesperforming a screen in pooled human immunoglobulins) (see, for example,FIG. 4).

In some embodiments, at the completion of both screens, variant AAVcapsids are chosen from each screen for full Sanger sequencing andphylogenetic comparisons to parental serotypes (i.e., parentalnon-variant capsid polypeptide sequences). In some embodiments, theparental non-variant capsid polypeptide sequences are those that wentinto the initial library. The most highly selected variants (forexample, those that exhibit the highest increase in transduction and/ortropism in human liver tissue or human hepatocyte cells and an enhancedneutralization profile) from the screens are isolated and vectorizedwith expression constructs, in some cases for use in subsequentvalidation experiments. In some embodiments, in order to assess thegenetic contribution of each parental AAV serotype (i.e., non-variantparent capsid polypeptide) to the evolved capsids (i.e., variant capsidpolypeptides) selected from each screen, crossover mapping can beperformed (see, for example, FIG. 5). Both methodologies demonstrate thehighly shuffled nature of the evolved capsid variants and highlightedboth unique and shared domains present in selected capsids.

In some embodiments, the parental capsids (i.e., non-variant parentcapsid polypeptides) that contribute the most to the evolved variantsinclude AAV2, and AAV3b. In some embodiments, the variant capsidpolypeptides comprise regions from AAV1, AAV2, AAV3b, AAV6, AAV8,AAV9_hu14 and de novo mutations. In some embodiments, no variants (i.e.,variant capsid polypeptide) have capsid fragment regions from AAV4, 5,bovine or avian. In some embodiments, diverse shuffling was achieved andmaintained along the length of Cap, including VP1, VP2 and VP3.

In vitro characterizations are used to demonstrate the significantincrease in transduction by variant capsid polypeptides over controlserotypes (i.e., non-variant parent capsid polypeptides) in variousliver-derived cell lines.

For such analyses, large-scale ultrapure productions of AAV vectorizedvariants (AAV vectors composed of variant capsid polypeptides) can becarried out and those capable of producing high titers sufficient foreventual clinical use (for example, variants AAV-NP84, AAV-NP59,AAV-NP40, and AAV-NP30) can be considered further for validation. Insome embodiments, in human liver cells or human hepatocyte cells,shuffled variants (i.e., variant capsid polypeptides) exhibitingsignificantly increased functional transduction can be selected by thepresent invention. In some embodiments, the variant AAV capsidpolypeptides further exhibit increased transduction or tropism in mousehepatocytes in vivo.

AAV Vector Elements

The nucleic acid insert (also referred to as a heterologous nucleotidesequence) can be operably linked to control elements directing thetranscription or expression thereof in the nucleotide sequence in vivo.Such control elements can comprise control sequences normally associatedwith the selected gene (e.g., endogenous cellular control elements).Alternatively, heterologous control sequences can be employed. Usefulheterologous control sequences generally include those derived fromsequences encoding mammalian or viral genes. Examples include, but arenot limited to, the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, an endogenous cellular promoterheterologous to the gene of interest, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, synthetic promoters, hybrid promoters, and thelike. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, can also be used. Such promoter sequencesare commercially available from, e.g., Stratagene (San Diego, Calif.).

In some embodiments, a cell type-specific or a tissue-specific promotercan be operably linked to nucleic acid insert (also referred to as aheterologous nucleotide sequence) encoding the heterologous geneproduct, and allowing for selectively or preferentially producing a geneproduct in a particular cell type(s) or tissue(s). In some embodiments,an inducible promoter can be operably linked to the heterologous nucleicacid.

In some embodiments, the nucleic acid is packaged with the variantcapsid polypeptides of the invention. In some embodiments, the nucleicacid insert or packaged nucleic acid is at least 3000 nucleic acids toat least 5000 nucleic acids. In some embodiments, the nucleic acidinsert or packaged nucleic acid is at least 3500 nucleic acids to atleast 4500 nucleic acids. In some embodiments, the nucleic acid insertor packaged nucleic acid is at least 4000 nucleic acids to at least 5000nucleic acids. In some embodiments, the nucleic acid insert or packagednucleic acid is at least 4200 nucleic acids to at least 4900 nucleicacids. In some embodiments, the nucleic acid insert or packaged nucleicacid is at least 4400 nucleic acids to at least 4800 nucleic acids. Insome embodiments, the nucleic acid insert or packaged nucleic acid is atleast about 4700 nucleic acids.

In some embodiments, the AAV vector packaged by the variant capsidpolypeptides is at least about 2000 nucleic acids in total length and upto about 5000 nucleic acids in total length. In some embodiments, theAAV vector packaged by the variant capsid polypeptides is about 2000nucleic acids, about 2400 nucleic acids, about 2800 nucleic acids, about3000 nucleic acids, about 3200 nucleic acids, about 3400 nucleic acids,about 3600 nucleic acids, about 3800 nucleic acids, about 4000 nucleicacids, about 4200 nucleic acids, about 4400 nucleic acids, about 4600nucleic acids, about 4700 nucleic acids, or about 4800 nucleic acids. Insome embodiments, the AAV vector packaged by the variant capsidpolypeptides is between about 2000 nucleic acids (2 kb) and about 5000nucleic acids (5 kb). In some embodiments, the AAV vector packaged bythe variant capsid polypeptides is between about 2400 nucleic acids (2.4kb) and about 4800 nucleic acids (4.8 kb). In some embodiments, the AAVvector packaged by the variant capsid polypeptides is between about 3000nucleic acids (3 kb) and about 5000 nucleic acids (5 kb). In someembodiments, the AAV vector packaged by the variant capsid polypeptidesis between about 3000 nucleic acids (3 kb) and about 4000 nucleic acids(4 kb).

The AAV vectors or AAV virions disclosed herein can also includeconventional control elements operably linked to the nucleic acid insert(also referred to as a heterologous nucleotide sequence) in a mannerpermitting transcription, translation and/or expression in a celltransfected with the AAV vector or infected with the AAV virion producedaccording to the present invention. As used herein, “operably linked”sequences include both expression control sequences that are contiguouswith the gene of interest and expression control sequences that act intrans or at a distance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters selected from native, constitutive,inducible and/or tissue-specific, are known in the art and may beutilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), theSV40 promoter, the dihydrofolate reductase promoter, the beta-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1promoter (Invitrogen). Inducible promoters allow regulation of geneexpression and can be regulated by exogenously supplied compounds,environmental factors such as temperature, or the presence of a specificphysiological state, e.g., acute phase, a particular differentiationstate of the cell, or in replicating cells only. Inducible promoters andinducible systems are available from a variety of commercial sources,including, without limitation, Invitrogen, Clonetech and Ariad. Manyother systems have been described and can be readily selected by one ofskill in the art. Examples of inducible promoters regulated byexogenously supplied compounds, include, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system(WO 98/10088); the ecdysone insect promoter (No et al., (1996) Proc.Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline-repressible system(Gossen et al., (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551), thetetracycline-inducible system (Gossen et al., (1995) Science,268:1766-1769, see also Harvey et al., (1998) Curr. Opin. Chem. Biol.,2:512-518), the RU486-inducible system (Wang et al., (1997) Nat.Biotech., 15:239-243 and Wang et al., (1997) Gene Ther., 4:432-441) andthe rapamycin-inducible system (Magari et al., (1997) J. Clin. Invest.,100:2865-2872). Other types of inducible promoters useful in thiscontext are those regulated by a specific physiological state, e.g.,temperature, acute phase, a particular differentiation state of thecell, or in replicating cells only.

In another embodiment, the native promoter for the nucleic acid insert(also referred to as a heterologous nucleotide sequence) will be used.The native promoter may be preferred when it is desired that expressionof the nucleic acid insert (also referred to as a heterologousnucleotide sequence) should mimic the native expression. The nativepromoter may be used when expression of the nucleic acid insert (alsoreferred to as a heterologous nucleotide sequence) must be regulatedtemporally or developmentally, or in a tissue-specific manner, or inresponse to specific transcriptional stimuli. In a further embodiment,other native expression control elements, such as enhancer elements,polyadenylation sites or Kozak consensus sequences may also be used tomimic the native expression.

Another embodiment of the nucleic acid insert (also referred to as aheterologous nucleotide sequence) includes a gene operably linked to atissue-specific promoter. For instance, if expression in liver tissue isdesired, a promoter active in liver tissue should be used. Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus corepromoter, Sandig et al., (1996) Gene Ther., 3:1002-9; alpha-fetoprotein(AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503-14), boneosteocalcin (Stein et al., (1997)Mol. Biol. Rep., 24:185-96); bonesialoprotein (Chen et al., (1996) J. Bone Miner. Res., 11:654-64),lymphocytes (CD2, Hansal et al., (1998) J. Immunol., 161:1063-8;immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., (1993) Cell.Mol. Neurobiol., 13:503-15), neurofilament light-chain gene (Piccioli etal., (1991) Proc. Natl. Acad. Sci. USA, 88:5611-5), and theneuron-specific vgf gene (Piccioli et al., (1995) Neuron, 15:373-84),among others.

In various embodiments, AAV vectors or AAV virions carrying one or moretherapeutically useful nucleic acid inserts (also referred to as aheterologous nucleotide sequence) also include selectable markers orreporter genes, e.g., sequences encoding geneticin, hygromycin orpuromycin resistance, among others. Selectable reporters or marker genescan be used to signal the presence of the plasmids/vectors in bacterialcells, including, for example, examining ampicillin resistance. Othercomponents of the plasmid may include an origin of replication.Selection of these and other promoters and vector elements areconventional and many such sequences are available (see, e.g., Sambrooket al., and references cited therein).

Host Cells and Packaging

Host cells are necessary for generating infectious AAV vectors as wellas for generating AAV virions based on the disclosed AAV vectors.Accordingly, the present invention provides host cells for generationand packaging of AAV virions based on the AAV vectors of the presentinvention. A variety of host cells are known in the art and find use inthe methods of the present invention. Any host cells described herein orknown in the art can be employed with the compositions and methodsdescribed herein.

The present invention provides host cells, e.g., isolated (geneticallymodified) host cells, comprising a subject nucleic acid. A subject hostcell can be an isolated cell, e.g., a cell in in vitro culture. Asubject host cell is useful for producing a subject AAV vector or AAVvirion, as described below. Where a subject host cell is used to producea subject AAV virion, it is referred to as a “packaging cell.” In someembodiments, a subject host cell is stably genetically modified with asubject AAV vector. In other embodiments, a subject host cell istransiently genetically modified with a subject AAV vector.

In some embodiments, a subject nucleic acid is introduced stably ortransiently into a host cell, using established techniques, including,but not limited to, electroporation, calcium phosphate transfection,liposome-mediated transfection, baculovirus infection, and the like. Forstable transformation, a subject nucleic acid will generally furtherinclude a selectable marker, e.g., any of several well-known selectablemarkers such as neomycin resistance, and the like.

Generally, when delivering the AAV vector according to the presentinvention by transfection, the AAV vector is delivered in an amount fromabout 5 μg to about 100 μg DNA, about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, or about 1×10⁵ cells. However, therelative amounts of vector DNA to host cells may be adjusted, takinginto consideration such factors as the selected vector, the deliverymethod and the host cells selected and such adjustments are within thelevel of skill of one in the art.

In some embodiments, the host cell for use in generating infectiousvirions can be selected from any biological organism, includingprokaryotic (e.g., bacterial) cells, and eukaryotic cells, including,insect cells, yeast cells and mammalian cells. A subject host cell isgenerated by introducing a subject nucleic acid (i.e., AAV vector) intoany of a variety of cells, e.g., mammalian cells, including, e.g.,murine cells, and primate cells (e.g., human cells). Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, CHO,293, Vero, NIH 3T3, PC12, Huh-7 Saos, C2C12, RAT1, L cells, HT1080,human embryonic kidney (HEK), human embryonic stem cells, human adulttissue stem cells, pluripotent stem cells, induced pluripotent stemcells, reprogrammed stem cells, organoid stem cells, bone marrow stemcells, HLHepG2, HepG2 and primary fibroblast, hepatocyte and myoblastcells derived from mammals including human, monkey, mouse, rat, rabbit,and hamster. The selection of the mammalian species providing the cellsis not a limitation of this invention; nor is the type of mammaliancell, i.e., fibroblast, hepatocyte, tumor cell, etc. The requirement forthe cell used is it is capable of infection or transfection by an AAVvector. In some embodiments, the host cell is one that has Rep and Capstably transfected in the cell, including in some embodiments a variantcapsid polypeptide as described herein. In some embodiments, the hostcell expresses a variant capsid polypeptide of the invention or part ofan AAV vector as described herein, such as a heterologous nucleic acidsequence contained within the AAV vector.

In some embodiments, the preparation of a host cell according to theinvention involves techniques such as assembly of selected DNAsequences. This assembly may be accomplished utilizing conventionaltechniques. Such techniques include cDNA and genomic cloning, which arewell known and are described in Sambrook et al., cited above, use ofoverlapping oligonucleotide sequences of the adenovirus and AAV genomes,combined with polymerase chain reaction, synthetic methods, and anyother suitable methods providing the desired nucleotide sequence.

In some embodiments, introduction of the AAV vector into the host cellmay also be accomplished using techniques known to the skilled artisanand as discussed throughout the specification. In a preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes providing trans-acting E1 proteins).

In some embodiments, a subject genetically modified host cell includes,in addition to a nucleic acid comprising a nucleotide sequence encodinga variant AAV capsid protein, as described above, a nucleic acid thatcomprises a nucleotide sequence encoding one or more AAV Rep(replication) proteins. In other embodiments, a subject host cellfurther comprises an AAV vector. An AAV virion can be generated using asubject host cell. Methods of generating an AAV virion are described in,e.g., U.S. Patent Publication No. 2005/0053922 and U.S. PatentPublication No. 2009/0202490.

In addition to the AAV vector, in exemplary embodiments, the host cellcontains the sequences driving expression of the AAV capsid polypeptide(including variant capsid polypeptides and non-variant parent capsidpolypeptides) in the host cell and Rep (replication) sequences of thesame serotype as the serotype of the AAV Inverted Terminal Repeats(ITRs) found in the nucleic acid insert (also referred to as aheterologous nucleotide sequence), or a cross-complementing serotype.The AAV capsid and Rep (replication) sequences may be independentlyobtained from an AAV source and may be introduced into the host cell inany manner known to one of skill in the art or as described herein.Additionally, when pseudotyping an AAV vector in an AAV8 capsid forexample, the sequences encoding each of the essential Rep (replication)proteins may be supplied by AAV8, or the sequences encoding the Rep(replication) proteins may be supplied by different AAV serotypes (e.g.,AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, and/or AAV9).

In some embodiments, the host cell stably contains the capsid proteinunder the control of a suitable promoter (including, for example, thevariant capsid polypeptides of the invention), such as those describedabove. In some embodiments, the capsid protein is expressed under thecontrol of an inducible promoter. In some embodiments, the capsidprotein is supplied to the host cell in trans. When delivered to thehost cell in trans, the capsid protein may be delivered via a plasmidcontaining the sequences necessary to direct expression of the selectedcapsid protein in the host cell. In some embodiments, when delivered tothe host cell in trans, the vector encoding the capsid protein(including, for example, the variant capsid polypeptides of theinvention) also carries other sequences required for packaging the AAV,e.g., the Rep (replication) sequences.

In some embodiments, the host cell stably contains the Rep (replication)sequences under the control of a suitable promoter, such as thosedescribed above. In some embodiments, the essential Rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the Rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the Rep proteins may bedelivered via a plasmid containing the sequences necessary to directexpression of the selected Rep proteins in the host cell. In someembodiments, when delivered to the host cell in trans, the vectorencoding the capsid protein (including, for example, the variant capsidpolypeptides of the invention) also carries other sequences required forpackaging the AAV vector, e.g., the Rep sequences.

In some embodiments, the Rep and capsid sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an unintegrated episome. In another embodiment, the Rep andcapsid sequences are stably integrated into the chromosome of the cell.Another embodiment has the Rep and capsid sequences transientlyexpressed in the host cell. For example, a useful nucleic acid moleculefor such transfection comprises, from 5′ to 3′, a promoter, an optionalspacer interposed between the promoter and the start site of the Repgene sequence, an AAV Rep gene sequence, and an AAV capsid genesequence.

Although the molecule(s) providing Rep and capsid can exist in the hostcell transiently (i.e., through transfection), in some embodiments, oneor both of the Rep and capsid proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of the invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above.

In some embodiments, the packaging host cell can require helperfunctions in order to package the AAV vector of the invention into anAAV virion. In some embodiments, these functions may be supplied by aherpesvirus. In some embodiments, the necessary helper functions areeach provided from a human or non-human primate adenovirus source, andare available from a variety of sources, including the American TypeCulture Collection (ATCC), Manassas, Va. (US). In some embodiments, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Insome embodiments, the host cell may contain other adenoviral genes suchas VAI RNA. In some embodiments, no other adenovirus genes or genefunctions are present in the host cell.

Heterologous Nucleic Acid, Nucleic Acid Gene Products, and PolypeptideGene Products

In various embodiments, the invention provides variant capsidpolypeptides capable of forming capsids capable of packaging a varietyof therapeutic molecules, including nucleic acids and polypeptides. Insome embodiments, the therapeutic molecule is a vaccine. In variousembodiments, the invention provides for AAV vectors capable ofcontaining nucleic acid inserts, including for example, transgeneinserts or other nucleic acid inserts. This allows for vectors capableof expressing polypeptides. Such nucleic acids can comprise heterologousnucleic acid, nucleic acid gene products, and polypeptide gene products.Features of the nucleic acid inserts are described below.

In some embodiments, the AAV vectors described herein contain nucleicacid inserts. In some embodiments, the nucleic acid insert includes butis not limited to nucleic acid sequences selected from the groupconsisting of a non-coding RNA, a protein coding sequence, an expressioncassette, a multi-expression cassette, a sequence for homologousrecombination, a genomic gene targeting cassette, and a therapeuticexpression cassette.

In some embodiments, the expression cassette is a CRISPR/CAS expressionsystem.

In some embodiments, a nucleic acid insert comprises a heterologousnucleic acid comprising a nucleotide sequence encoding a heterologousgene product, e.g., a nucleic acid gene product or a polypeptide geneproduct. In some embodiments, the gene product is an interfering RNA(e.g., shRNA, siRNA, miRNA). In some embodiments, the gene product is anaptamer. The gene product can be a self-complementary nucleic acid. Insome embodiments, the gene product is a polypeptide.

Suitable heterologous gene product includes interfering RNA, antisenseRNA, ribozymes, and aptamers. Where the gene product is an interferingRNA (RNAi), suitable RNAi include RNAi that decrease the level of atarget polypeptide in a cell.

In some embodiments, exemplary polypeptides, nucleic acids, or othertherapeutic molecules include those useful in the treatment of liverdiseases and disorders. Liver disease and disorders include but are notlimited to any conditions that stop the liver from functioning properlyor prevent it from functioning well (i.e., functioning at normallevels). Symptoms of liver diseases and disorders can include but arenot limited to abdominal pain, yellowing of the skin or eyes (jaundice),abnormal results of liver function tests, liver fattening (including,for example, disproportional fattening), and cirrhosis of the liver.Liver diseases and disorders further include but are not limited toamebic liver abscess, autoimmune liver diseases and disorders(including, for example, autoimmune hepatitis, primary biliary cirrhosis(PBC), and primary sclerosing cholangitis (PSC)), biliary atresia,cirrhosis, coccidioidomycosis, delta agent (hepatitis D), drug-inducedcholestasis, hemochromatosis, viral hepatitis (including, for example,Hepatitis A, Hepatitis B, and Hepatitis C), neonatal hepatitis,hepatocellular carcinoma (HCC, as well as other liver cancer),fibrolamellar hepatocellular carcinoma, liver disease due to alcohol,pyogenic liver abscess, Reye's syndrome, Sclerosing cholangitis,Wilson's disease, acute liver failure (caused by, for example, drugs,toxins, and various other diseases), alcoholic liver disease,autoimmune-associated diseases, Budd-Chiari syndrome, hypercoagulabledisorders, parasitic infection, chronic bile duct obstruction(including, for example, due to tumors, gallstones, inflammation, andtrauma), hemochromatosis, alpha-1 antitrypsin (A1A) deficiency, Wilsondisease, Alagille Syndrome, cystic disease of the liver, galactosemia,Gilbert's Syndrome, hemochromatosis, liver disease in pregnancy,Lysosomal Acid Lipase Deficiency (LALD), porphyria, sarcoidosis, Type 1Glycogen Storage Disease, Tyrosinemia, Alveolar hydatid disease,bacillary peliosis, congenital hepatic fibrosis, congestive hepatopathy,gastric antral vascular ectasia, hepatic encephalopathy,hepatolithiasis, hepatopulmonary syndrome, hepatorenal syndrome,hepatosplenomegaly, hepatotoxicity, Indian childhood cirrhosis,Laennec's cirrhosis, Lyngstadaas Syndrome, peliosis hepatis, progressivefamilial intrahepatic cholestasis, Zahn infarct, Zieve's syndrome, andnonalcoholic fatty liver disease (NAFLD).

In some embodiments, exemplary polypeptides, nucleic acids, or othertherapeutic molecules include those useful in the treatment of raresarcoglycanopathies and dystrophinopathies like Duchenne musculardystrophy, limb girdle muscle disease, and spinal muscular atrophy, aswell as other muscle tissue related diseases. Exemplary muscle tissuerelated diseases include but are not limited to Acid Maltase Deficiency(AMD), Amyotrophic Lateral Sclerosis (ALS), Andersen-Tawil Syndrome,Becker Muscular Dystrophy (BMD), Becker Myotonia Congenita, BethlemMyopathy, Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy),Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency (CPTDeficiency), Central Core Disease (CCD), Centronuclear Myopathy,Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD),Congenital Myasthenic Syndromes (CMS), Congenital Myotonic Dystrophy,Cori Disease (Debrancher Enzyme Deficiency), Debrancher EnzymeDeficiency, Dejerine-Sottas Disease (DSD), Dermatomyositis (DM), DistalMuscular Dystrophy (DD), Duchenne Muscular Dystrophy (DMD), DystrophiaMyotonica (Myotonic Muscular Dystrophy), Emery-Dreifuss MuscularDystrophy (EDMD), Endocrine Myopathies, Eulenberg Disease (ParamyotoniaCongenita), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD),Finnish (Tibial) Distal Myopathy, Forbes Disease (Debrancher EnzymeDeficiency), Friedreich's Ataxia (FA), Fukuyama Congenital MuscularDystrophy, Glycogenosis Type 10, Glycogenosis Type 11, Glycogenosis Type2, Glycogenosis Type 3, Glycogenosis Type 5, Glycogenosis Type 7,Glycogenosis Type 9, Gowers-Laing Distal Myopathy, Hauptmann-ThanheuserMD (Emery-Dreifuss Muscular Dystrophy), Hereditary Inclusion-BodyMyositis, Hereditary Motor and Sensory Neuropathy (Charcot-Marie-ToothDisease), Hyperthyroid Myopathy, Hypothyroid Myopathy, Inclusion-BodyMyositis (IBM), Inherited Myopathies, Integrin-Deficient CongenitalMuscular Dystrophy, Kennedy Disease (Spinal-Bulbar Muscular Atrophy),Kugelberg-Welander Disease (Spinal Muscular Atrophy), LactateDehydrogenase Deficiency, Lambert-Eaton Myasthenic Syndrome (LEMS),Limb-Girdle Muscular Dystrophy (LGMD), Lou Gehrig's Disease (AmyotrophicLateral Sclerosis), McArdle Disease (Phosphorylase Deficiency),Merosin-Deficient Congenital Muscular Dystrophy, Metabolic Diseases ofMuscle, Mitochondrial Myopathy, Miyoshi Distal Myopathy, Motor NeuroneDisease, Muscle-Eye-Brain Disease, Myasthenia Gravis (MG), MyoadenylateDeaminase Deficiency, Myofibrillar Myopathy, MyophosphorylaseDeficiency, Myotonia Congenita (MC), Myotonic Muscular Dystrophy (MMD),Myotubular Myopathy (MTM or MM), Nemaline Myopathy, Nonaka DistalMyopathy, Oculopharyngeal Muscular Dystrophy (OPMD), ParamyotoniaCongenita, Pearson Syndrome, Periodic Paralysis, Peroneal MuscularAtrophy (Charcot-Marie-Tooth Disease), Phosphofructokinase Deficiency,Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency,Phosphorylase Deficiency, Phosphorylase Deficiency, Polymyositis (PM),Pompe Disease (Acid Maltase Deficiency), Progressive ExternalOphthalmoplegia (PEO), Rod Body Disease (Nemaline Myopathy), SpinalMuscular Atrophy (SMA), Spinal-Bulbar Muscular Atrophy (SBMA), SteinertDisease (Myotonic Muscular Dystrophy), Tarui Disease(Phosphofructokinase Deficiency), Thomsen Disease (Myotonia Congenita),Ullrich Congenital Muscular Dystrophy, Walker-Warburg Syndrome(Congenital Muscular Dystrophy), Welander Distal Myopathy,Werdnig-Hoffmann Disease (Spinal Muscular Atrophy), and ZASP-RelatedMyopathy.

In some embodiments, exemplary polypeptides include neuroprotectivepolypeptides and anti-angiogenic polypeptides. Suitable polypeptidesinclude, but are not limited to, glial derived neurotrophic factor(GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliaryneurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growthfactor-.beta.), brain derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growthfactor (EGF), pigment epithelium derived factor (PEDF), a Wntpolypeptide, soluble Flt-1, angiostatin, endostatin, VEGF, an anti-VEGFantibody, a soluble VEGFR, Factor VIII (FVIII), Factor IX (FIX), and amember of the hedgehog family (sonic hedgehog, Indian hedgehog, anddesert hedgehog, etc.).

In some embodiments, useful therapeutic products encoded by theheterologous nucleic acid sequence include hormones and growth anddifferentiation factors including, without limitation, insulin,glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormonereleasing factor (GRF), follicle stimulating hormone (FSH), luteinizinghormone (LH), human chorionic gonadotropin (hCG), vascular endothelialgrowth factor (VEGF), angiopoietins, angiostatin, granulocyte colonystimulating factor (GCSF), erythropoietin (EPO), connective tissuegrowth factor (CTGF), basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin growth factors I and II(IGF-I and IGF-II), any one of the transforming growth factor alphasuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

In some embodiments, useful heterologous nucleic acid sequence productsinclude proteins that regulate the immune system including, withoutlimitation, cytokines and lymphokines such as thrombopoietin (TPO),interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12 andIL-18), monocyte chemoattractant protein, leukemia inhibitory factor,granulocyte-macrophage colony stimulating factor, Fas ligand, tumornecrosis factors alpha and .beta., interferons .alpha., .beta., and.gamma., stem cell factor, flk-2/flt3 ligand. Gene products produced bythe immune system are also useful in the present invention. Theseinclude, without limitations, immunoglobulins IgG, IgM, IgA, IgD andIgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

In some embodiments, useful heterologous nucleic acid sequence productsinclude any one of the receptors for the hormones, growth factors,cytokines, lymphokines, regulatory proteins and immune system proteins.Useful heterologous nucleic acid sequence s also include receptors forcholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses the use of geneproducts such as members of the steroid hormone receptor superfamilyincluding glucocorticoid receptors and estrogen receptors, Vitamin Dreceptors and other nuclear receptors. In addition, useful gene productsinclude transcription factors such as jun, fos, max, mad, serum responsefactor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containingproteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZFS, NFAT, CREB, HNF-4,C/EBP, SP1, CCAAT-box binding proteins, interferon regulation factor(IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-boxbinding proteins, e.g., GATA-3, and the forkhead family of winged helixproteins.

In some embodiments, useful heterologous nucleic acid sequence productsinclude, carbamoyl synthetase I, ornithine transcarbamylase,arginosuccinate synthetase, arginosuccinate lyase, arginase,fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence. Still other useful gene products includeenzymes useful in enzyme replacement therapy, and which are useful in avariety of conditions resulting from deficient activity of enzyme. Forexample, enzymes containing mannose-6-phosphate may be utilized intherapies for lysosomal storage diseases (e.g., a suitable gene includesthat encoding β-glucuronidase (GUSB)).

In some embodiments, useful heterologous nucleic acid sequence productsinclude those used for treatment of hemophilia, including hemophilia B(including Factor IX) and hemophilia A (including Factor VIII and itsvariants, such as the light chain and heavy chain of the heterodimer andthe B-deleted domain; U.S. Pat. Nos. 6,200,560 and 6,221,349). TheFactor VIII gene codes for 2351 amino acids and the protein has sixdomains, designated from the amino to the terminal carboxy terminus asA1-A2-B-A3-C1-C2 (Wood et al., (1984) Nature, 312:330; Vehar et al.,(1984) Nature 312:337; and Toole et al., (1984) Nature, 342:337). HumanFactor VIII is processed within the cell to yield a heterodimerprimarily comprising a heavy chain containing the A1, A2 and B domainsand a light chain containing the A3, C1 and C2 domains. Both the singlechain polypeptide and the heterodimer circulate in the plasma asinactive precursors, until activated by thrombin cleavage between the A2and B domains, releasing the B domain and results in a heavy chainconsisting of the A1 and A2 domains. The B domain is deleted in theactivated procoagulant form of the protein. Additionally, in the nativeprotein, two polypeptide chains (“a” and “b”), flanking the B domain,are bound to a divalent calcium cation.

In some embodiments, useful gene products include non-naturallyoccurring polypeptides, such as chimeric or hybrid polypeptides having anon-naturally occurring amino acid sequence containing insertions,deletions or amino acid substitutions. For example, single-chainengineered immunoglobulins could be useful in certain immunocompromisedpatients. Other types of non-naturally occurring gene sequences includeantisense molecules and catalytic nucleic acids, such as ribozymes, usedto reduce overexpression of a target.

In some embodiments, the present invention provides methods fortreatment of a stem cell disorder, for example a disorder in either bonemarrow stem cells or adult tissue stem cells (i.e., somatic stem cells).In some embodiments, adult stem cells can include organoid stem cells(i.e., stem cells derived from any organ or organ system of interestwithin the body). Organs of the body include for example but are notlimited to skin, hair, nails, sense receptors, sweat gland, oil glands,bones, muscles, brain, spinal cord, nerve, pituitary gland, pinealgland, hypothalamus, thyroid gland, parathyroid, thymus, adrenals,pancreas (islet tissue), heart, blood vessels, lymph nodes, lymphvessels, thymus, spleen, tonsils, nose, pharynx, larynx, trachea,bronchi, lungs, mouth, pharynx, esophagus, stomach, small intestine,large intestine, rectum, anal canal, teeth, salivary glands, tongue,liver, gallbladder, pancreas, appendix, kidneys, ureters, urinarybladder, urethra, testes, ductus (vas) deferens, urethra, prostate,penis, scrotum, ovaries, uterus, uterine (fallopian) tubes, vagina,vulva, and mammary glands (breasts). Organ systems of the body includebut are not limited to the integumentary system, skeletal system,muscular system, nervous system, endocrine system, cardiovascularsystem, lymphatic system, respiratory system, digestive system, urinarysystem, and reproductive system. In some embodiments, the disorder fortreatment is a disorder in any one or more organoid stem cells (i.e.,stem cells derived from any organ or organ system of interest within thebody). In some embodiments, the treatment is in vivo (for example,administration of the variant capsid polypeptides is directly to thesubject). In some embodiments, the treatment is ex vivo (for example,administration of the variant capsid polypeptides is to stem cellsisolated from the subject and the treated stem cells are then returnedto the subject).

Reduction and/or modulation of expression of a heterologous nucleic acidsequence is particularly desirable for treatment of hyperproliferativeconditions characterized by hyperproliferating cells, such as cancersand psoriasis. Target polypeptides include those polypeptides producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include polypeptides encoded byoncogenes such as myb, myc, fyn, and the translocation gene bcr/abl,ras, src, P53, neu, trk and EGRF. In addition to oncogene products astarget antigens, target polypeptides for anti-cancer treatments andprotective regimens include variable regions of antibodies made by Bcell lymphomas and variable regions of T cell receptors of T celllymphomas which, in some embodiments, are used as target antigens forautoimmune disease. Other tumor-associated polypeptides can be used astarget polypeptides such as polypeptides found at higher levels in tumorcells including the polypeptide recognized by monoclonal antibody 17-1Aand folate binding polypeptides.

In some embodiments, suitable therapeutic polypeptides and proteinsinclude those useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells producing “self”-directed antibodies. T cellmediated autoimmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

In some embodiments, heterologous nucleic acid sequences encode forimmunogens useful to immunize (i.e., useful as, for example, a vaccine)a human or non-human animal against other pathogens including bacteria,viruses, fungi, parasitic microorganisms or multicellular parasitesinfecting human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci (and the toxinsproduced thereby, e.g., enterotoxin B); and streptococci. Pathogenicgram-negative cocci include meningococcus; gonococcus. Pathogenicenteric gram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (causes chancroid); brucella species(brucellosis); Francisella tularensis (causes tularemia); Yersiniapestis (plague) and other Yersinia (pasteurella); Streptobacillusmoniliformis and spirillum; Gram-positive bacilli include Listeriamonocytogenes; Erysipelothrix rhusiopathiae; Corynebacterium diphtheria(causes diphtheria); cholera; Bacillus. anthracia (causes anthrax);donovanosis (granuloma inguinale; caused by Klebsiella granulomatis);and bartonellosis. Diseases caused by pathogenic anaerobic bacteriainclude tetanus; botulism (Clostridum botulinum and its toxin);Clostridium perfringens and its epsilon toxin; other clostridia;tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetaldiseases include syphilis; treponematoses: yaws, pinta and endemicsyphilis; and leptospirosis. Other infections caused by higher pathogenbacteria and pathogenic fungi include glanders (Burkholderia mallei);actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever; Rocky Mountain spottedfever; Q fever (Coxiella burnetti); and Rickettsialpox. Examples ofmycoplasma and chlamydial infections include: Mycoplasma pneumoniae;lymphogranuloma venereum (caused by Chlamydia trachomatis); psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompassingpathogenic protozoans and helminths and infections produced therebyinclude: amebiasis (caused by Entamoeba histolytica); malaria (caused byPlasmodium); Leishmaniasis (caused by Leishmania); trypanosomiasis(caused by Trypanosoma); toxoplasmosis (caused by Toxoplasma gondii);Pneumocystis carinii; babesiosis (caused by Babesia); giardiasis (causedby Giardia lamblia); trichinosis (caused by roundworms of the genusTrichinella); filariasis (caused by roundworms of Filarioidea);schistosomiasis (carried by fresh water snails infected with one of thefive varieties of the parasite Schistosoma); nematodes (Nematoda);trematodes or flukes (Platyhelminthes); and cestode (Cestoidea;tapeworm) infections. Examples of viruses include but are not limited tohuman immunodeficiency virus (HIV; e.g., HIV-1 and HIV-2), influenza(e.g., influenza A, influenza B, and influenza C), parainfluenzahepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitisD, and hepatitis E), herpes viruses (HSV; HHV; e.g., herpes virus types1, 2, 3, 4, 5, 6A, 6B, 7, and 8, including herpes simplex virus types 1and 2, aka, HSV-1; HSV-2), varicella-zoster virus (HHV-3), Epstein Barrvirus (HHV-4), Roseolovirus (HHV-6A and HHV-6B); Rous sarcoma virus,cytomegalovirus (HHV-5), Kaposi's sarcoma-associated herpesvirus; KSHV;HHV-8), papovirus (e.g., human papilloma virus; HPV; HPV-1, HPV-2,HPV-16, and HPV-18), parvovirus (e.g., Parvovirus B19), orthomyxovirus,paramyxovirus (e.g., morbillivirus, respirovirus, rubulavirus,ferlavirus, pneumovirus, and metapneumovirus), picornavirus (e.g.,foot-and-mouth disease virus, aquamavirus A, encephalomyocarditis virus,theilovirus, cosavirus A, cadicivirus A, enterovirus A, enterovirus B,enterovirus C, enterovirus D, enterovirus E, enterovirus F, enterovirusG, enterovirus H, enterovirus J, rhinovirus A, rhinovirus B, rhinovirusC, aichivirus A, aichivirus B, aichivirus C, melegrivirus A, humanparechovirus, ljungan virus, and salivirus A), togavirus (e.g.,flavivirus, alphavirus, and rubivirus), Cowpox virus, Horsepox virus,Crimean-Congo hemorrhagic fever virus, Dengue virus, Eastern equineencephalitis virus, Hantaan virus, Human coronavirus, Human enterovirus68, Human enterovirus 70, non-HIV retroviruses, rhinovirus, respiratorysyncytial virus (RSV), SARS coronavirus, Human spumaretrovirus, HumanT-lymphotropic virus, Isfahan virus, Japanese encephalitis virus, Lassavirus, Lymphocytic choriomeningitis virus, MERS coronavirus, measlesvirus, Mengo encephalomyocarditis virus, Monkeypox virus, mumps virus,Norwalk virus, Pichinde virus, Poliovirus, Rabies virus, rotavirus(e.g., rotavirus A, rotavirus B, and rotavirus C), Rubella virus, St.Louis encephalitis virus, Toscana virus, Uukuniemi virus, Venezuelanequine encephalitis virus, Western equine encephalitis virus, West Nilevirus, Yellow fever virus, and Ebola, as well as any other viruses knownto those of skill in the art.

Methods for Generating an AAV Virion

In various embodiments, the invention provides a method for generatingan AAV virion of the invention. A variety of methods of generating AAVvirions are known in the art and can be used to generate AAV virionscomprising the AAV vectors described herein. Generally, the methodsinvolved inserting or transducing an AAV vector of the invention into ahost cell capable of packaging the AAV vector into and AAV virion.Exemplary methods are described and referenced below; however, anymethod known to one of skill in the art can be employed to generate theAAV virions of the invention.

An AAV vector comprising a heterologous nucleic acid and used togenerate an AAV virion can be constructed using methods that are wellknown in the art. See, e.g., Koerber et al. (2009) Mol. Ther., 17:2088;Koerber et al. (2008) Mol. Ther., 16: 1703-1709; as well as U.S. Pat.Nos. 7,439,065, 6,951,758, and 6,491,907. For example, the heterologoussequence(s) can be directly inserted into an AAV genome with the majorAAV open reading frames (“ORFs”) excised therefrom. Other portions ofthe AAV genome can also be deleted, so long as a sufficient portion ofthe ITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (publishedMar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Curr. Topics Microbiol. Immunol. 158:97-129; Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994)Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.179:1867-1875.

In order to produce AAV virions, an AAV vector is introduced into asuitable host cell using known techniques, such as by transfection. Anumber of transfection techniques are generally known in the art. See,e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories,New York, Davis et al. (1986) Basic Methods in Molecular Biology,Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitabletransfection methods include calcium phosphate co-precipitation (Grahamet al. (1973) Virol. 52:456-467), direct micro-injection into culturedcells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome-mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

Suitable host cells for producing AAV virions include microorganisms,yeast cells, insect cells, and mammalian cells, that can be, or havebeen, used as recipients of a heterologous DNA molecule. The termincludes the progeny of the original cell transfected. Thus, a “hostcell” as used herein generally refers to a cell transfected with anexogenous DNA sequence. Cells from the stable human cell line, 293(readily available through, e.g., the American Type Culture Collectionunder Accession Number ATCC CRL1573) can be used. For example, the humancell line 293 is a human embryonic kidney cell line that has beentransformed with adenovirus type-5 DNA fragments (Graham et al. (1977)J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1b genes(Aiello et al. (1979) Virology 94:460). The 293 cell line is readilytransfected, and provides a convenient platform in which to produce AAVvirions.

Methods of producing an AAV virion in insect cells are known in the art,and can be used to produce a subject AAV virion. See, e.g., U.S. PatentPublication No. 2009/0203071; U.S. Pat. No. 7,271,002; and Chen (2008)Mol. Ther. 16:924.

In some embodiments, the AAV virion or AAV vector is packaged into aninfectious virion or virus particle, by any of the methods describedherein or known in the art.

In some embodiments, the variant capsid polypeptide allows for similarpackaging as compared to a non-variant parent capsid polypeptide.

In some embodiments, an AAV vector packaged with the variant capsidpolypeptide is transduced into cells in vivo better than a vectorpackaged with a non-variant parent capsid polypeptide.

In some embodiments, the AAV vector packaged with the variant capsidpolypeptide is transduced into cells in vitro better than a vectorpackaged with a non-variant parent capsid polypeptide.

In some embodiments, the variant capsid polypeptide results in nucleicacid expression higher than a nucleic acid packaged with a non-variantparent capsid polypeptide.

In some embodiments, the AAV vector packaged with said variant capsidpolypeptide results in transgene expression better than a transgenepackaged with a non-variant parent capsid polypeptide.

Pharmaceutical Compositions & Dosing

The present invention provides pharmaceutical compositions useful intreating subjects according to the methods of the invention as describedherein. Further, the present invention provides dosing regimens foradministering the described pharmaceutical compositions. The presentinvention provides pharmaceutical compositions comprising: a) a subjectAAV vector or AAV virion, as described herein as well as therapeuticmolecules packaged by or within capsids comprising variant polypeptidesas described herein; and b) a pharmaceutically acceptable carrier,diluent, excipient, or buffer. In some embodiments, the pharmaceuticallyacceptable carrier, diluent, excipient, or buffer is suitable for use ina human.

Such excipients, carriers, diluents, and buffers include anypharmaceutical agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, glycerol and ethanol. Pharmaceuticallyacceptable salts can be included therein, for example, mineral acidsalts such as hydrochlorides, hydrobromides, phosphates, sulfates, andthe like; and the salts of organic acids such as acetates, propionates,malonates, benzoates, and the like. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present in such vehicles. A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro, (2000) Remington: The Science and Practice ofPharmacy, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds.,7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

A subject composition can comprise a liquid comprising a subject variantAAV capsid polypeptide of the invention or AAV virion comprising avariant capsid polypeptide in solution, in suspension, or both. As usedherein, liquid compositions include gels. In some cases, the liquidcomposition is aqueous. In some embodiments, the composition is an insitu gellable aqueous composition, e.g., an in situ gellable aqueoussolution. Aqueous compositions have opthalmically compatible pH andosmolality.

Such compositions include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration or delivery, as set forth herein orknown to one of skill in the art. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byvarious routes.

Compositions suitable for parenteral administration comprise aqueous andnon-aqueous solutions, suspensions or emulsions of the active compound.Preparations are typically sterile and can be isotonic with the blood ofthe intended recipient. Non-limiting illustrative examples includewater, saline, dextrose, fructose, ethanol, animal, vegetable orsynthetic oils.

For transmucosal or transdermal administration (e.g., topical contact),penetrants can be included in the pharmaceutical composition. Penetrantsare known in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.For transdermal administration, the active ingredient can be formulatedinto aerosols, sprays, ointments, salves, gels, or creams as generallyknown in the art. For contact with skin, pharmaceutical compositionstypically include ointments, creams, lotions, pastes, gels, sprays,aerosols, or oils. Useful carriers include Vaseline®, lanolin,polyethylene glycols, alcohols, transdermal enhancers, and combinationsthereof.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Pharmaceutical compositions and delivery systems appropriate for the AAVvector or AAV virion and methods and uses of are known in the art (see,e.g., Remington: The Science and Practice of Pharmacy (2003) 20^(th)ed., Mack Publishing Co., Easton, Pa.; Remington's PharmaceuticalSciences (1990) 18^(th) ed., Mack Publishing Co., Easton, Pa.; The MerckIndex (1996) 12^(th) ed., Merck Publishing Group, Whitehouse, N.J.;Pharmaceutical Principles of Solid Dosage Forms (1993), TechnonicPublishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, PharmaceuticalCalculations (2001) 11^(th) ed., Lippincott Williams & Wilkins,Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

Doses can vary and depend upon whether the treatment is prophylactic ortherapeutic, the type, onset, progression, severity, frequency,duration, or probability of the disease treatment is directed to, theclinical endpoint desired, previous or simultaneous treatments, thegeneral health, age, gender, race or immunological competency of thesubject and other factors that will be appreciated by the skilledartisan. The dose amount, number, frequency or duration may beproportionally increased or reduced, as indicated by any adverse sideeffects, complications or other risk factors of the treatment or therapyand the status of the subject. The skilled artisan will appreciate thefactors that may influence the dosage and timing required to provide anamount sufficient for providing a therapeutic or prophylactic benefit.

Methods and uses of the invention as disclosed herein can be practicedwithin about 1 hour to about 2 hours, about 2 hours to about 4 hours,about 4 hours to about 12 hours, about 12 hours to about 24 hours orabout 24 hours to about 72 hours after a subject has been identified ashaving the disease targeted for treatment, has one or more symptoms ofthe disease, or has been screened and is identified as positive as setforth herein even though the subject does not have one or more symptomsof the disease. In some embodiments, the invention as disclosed hereincan be practiced within about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 24 hours, about 36 hours, about 48 hours, or about 72 hours ormore. Of course, methods and uses of the invention can be practicedabout 1 day to about 7 days, about 7 days to about 14 days, about 14days to about 21 days, about 21 days to about 48 days or more, months oryears after a subject has been identified as having the disease targetedfor treatment, has one or more symptoms of the disease, or has beenscreened and is identified as positive as set forth herein. In someembodiments, the invention as disclosed herein can be practiced withinabout 1 day, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 14 days, about 21 days, about 36days, or about 48 days or more.

In some embodiments, the present invention provides kits with packagingmaterial and one or more components therein. A kit typically includes alabel or packaging insert including a description of the components orinstructions for use in vitro, in vivo, or ex vivo, of the componentstherein. A kit can contain a collection of such components, e.g., avariant AAV capsid polypeptide, an AAV vector, or AAV virion andoptionally a second active, such as another compound, agent, drug orcomposition.

A kit refers to a physical structure housing one or more components ofthe kit. Packaging material can maintain the components sterilely, andcan be made of material commonly used for such purposes (e.g., paper,corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingthe manufacturer, lot numbers, manufacturer location and date, andexpiration dates. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.Labels or inserts can include information on a disease a kit componentmay be used for. Labels or inserts can include instructions for theclinician or subject for using one or more of the kit components in amethod, use, or treatment protocol or therapeutic regimen. Instructionscan include dosage amounts, frequency or duration, and instructions forpracticing any of the methods, uses, treatment protocols or prophylacticor therapeutic regimes described herein.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, complications or reactions, such as warnings to the subject orclinician regarding situations where it would not be appropriate to usea particular composition. Adverse side effects or complications couldalso occur when the subject has, will be or is currently taking one ormore other medications that may be incompatible with the composition, orthe subject has, will be or is currently undergoing another incompatibletreatment protocol or therapeutic regimen and, therefore, instructionscould include information regarding such incompatibilities.

Labels or inserts include “printed matter,” e.g., paper or cardboard, orseparate or affixed to a component, a kit or packing material (e.g., abox), or attached to an ampule, tube or vial containing a kit component.Labels or inserts can additionally include a computer readable medium,such as a bar-coded printed label, a disk, optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards.

Method of Treating a Disease

The present invention also provides methods for treatment of disease ina subject by administering the AAV vectors and/or nucleic acids of thepresent invention, where AAV vectors and/or nucleic acids describedherein packaged within a functional AAV capsid, wherein the functionalAAV capsid comprises one or more variant capsid polypeptides of thepresent invention. In an exemplary embodiment, the invention provides amethod of administering a pharmaceutical composition of the invention toa subject in need thereof to treat a disease of a subject. In variousembodiments, the subject is not otherwise in need of administration of acomposition of the invention. In some embodiments, the inventionprovides methods for vaccine administration.

In some embodiments, the variant AAV capsid polypeptide packages atherapeutic expression cassette comprised of a heterologous nucleic acidcomprising a nucleotide sequence encoding a heterologous gene product,such as for example a therapeutic protein or vaccine. In someembodiments, the AAV virion or AAV vector comprises a therapeuticexpression cassette comprised of a heterologous nucleic acid comprisinga nucleotide sequence encoding a heterologous gene product, such as forexample a therapeutic protein or vaccine.

In some embodiments, the variant capsid polypeptides of the inventionare employed as part of vaccine delivery. Vaccine delivery can includedelivery of any of the therapeutic proteins as well as nucleic acidsdescribed herein. In some embodiments, variant capsid polypeptides ofthe invention are employed as part of a vaccine regimen and dosedaccording to the methods described herein.

In some embodiments, the variant AAV capsid polypeptides, the AAVvirions, or AAV vectors of the invention are used in a therapeutictreatment regimen.

In some embodiments, the variant AAV capsid polypeptides, the AAVvirions, or AAV vectors of the invention are used for therapeuticpolypeptide production.

In some cases, a subject variant AAV capsid polypeptide or AAV vector,when introduced into the cells of a subject provides for high-levelproduction of the heterologous gene product packaged by the variant AAVcapsid polypeptide or encoded by the AAV. For example, a heterologouspolypeptide packaged by the variant AAV capsid polypeptide or encoded bythe AAV can be produced at a level of from about 1 μg to about 50 μg ormore.

In some cases, a subject variant AAV capsid polypeptide, AAV virion, orAAV vector, when introduced into a subject provides for production ofthe heterologous gene product packaged by the variant AAV capsidpolypeptide or encoded by the AAV vector in at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 50% at leastabout 60%, at least about 70%, at least about 80%, or more than 80%, ofthe target cells.

In some embodiments, the present invention provides a method of treatinga disease, the method comprising administering to an individual in needthereof an effective amount of a therapeutic molecule packaged by thevariant AAV capsid polypeptide or subject AAV virion as described above.

A variant AAV capsid polypeptide or subject AAV virion can beadministered systemically, regionally or locally, or by any route, forexample, by injection, infusion, orally (e.g., ingestion or inhalation),or topically (e.g., transdermally). Such delivery and administrationinclude intravenously, intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, intracranially,transdermally (topical), parenterally, e.g. transmucosally or rectally.Exemplary administration and delivery routes include intravenous (i.v.),intraperitoneal (i.p.), intrarterial, intramuscular, parenteral,subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal,parenterally, e.g. transmucosal, intra-cranial, intra-spinal, oral(alimentary), mucosal, respiration, intranasal, intubation,intrapulmonary, intrapulmonary instillation, buccal, sublingual,intravascular, intrathecal, intracavity, iontophoretic, intraocular,ophthalmic, optical, intraglandular, intraorgan, and intralymphatic.

In some cases, a therapeutically effective amount of a therapeuticmolecule packaged by the variant AAV capsid polypeptide or a subject AAVvirion is an amount that, when administered to an individual in one ormore doses, is effective to slow the progression of the disease ordisorder in the individual, or is effective to ameliorate symptoms. Forexample, a therapeutically effective amount of a therapeutic moleculepackaged by the variant AAV capsid polypeptide or a subject AAV virioncan be an amount that, when administered to an individual in one or moredoses, is effective to slow the progression of the disease by at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than about 80%, compared to theprogression of the disease in the absence of treatment with thetherapeutic molecule packaged by the variant AAV capsid polypeptide orthe AAV virion.

A therapeutic or beneficial effect of treatment is therefore anyobjective or subjective measurable or detectable improvement or benefitprovided to a particular subject. A therapeutic or beneficial effect canbut need not be complete ablation of all or any particular adversesymptom, disorder, illness, or complication of a disease. Thus, asatisfactory clinical endpoint is achieved when there is an incrementalimprovement or a partial reduction in an adverse symptom, disorder,illness, or complication caused by or associated with a disease, or aninhibition, decrease, reduction, suppression, prevention, limit orcontrol of worsening or progression of one or more adverse symptoms,disorders, illnesses, or complications caused by or associated with thedisease, over a short or long duration (hours, days, weeks, months,etc.).

Improvement of clinical symptoms can also be monitored by one or moremethods known to the art, and used as an indication of therapeuticeffectiveness. Clinical symptoms may also be monitored by any meansknown by those of skill in the art, including for example measure ofliver effectiveness, including for example but not limited to liverenzyme tests, MRI, bile acid profiling, or any other tests known to beuseful in examinting and/or determining liver function and/or a defectin liver function. Liver enzyme tests (sometimes refered to as liverfunction tests, or LFTs) include but are not limited to testing theblood for levels of aspartate aminotransferase (AST or SGOT), alanineaminotransferase (ALT or SGPT), alkaline phosphatase (ALP), 5′nucleotidase, gamma-glutamyl transpeptidase (GGT), bilirubin, albumin,and alpha-1 antitrypsin (A1A). Prothrombin time (PT; clotting time;often expressed as international normalized ratio (INR)) can also bemeasured, due to the liver's involvement in clotting factor production.Bilirubin levels can also be measured in the urine as a test for liverfunction.

In some embodiments, a therapeutic molecule (including, for example, avaccine) packaged by the variant AAV capsid polypeptide, a subject AAVvirion, or AAV virus, when introduced into a subject, provides forproduction of the heterologous gene product for a period of time of fromabout 2 days to about 6 months, e.g., from about 2 days to about 7 days,from about 1 week to about 4 weeks, from about 1 month to about 2months, or from about 2 months to about 6 months. In some embodiments,therapeutic molecule (including, for example, a vaccine) packaged by thevariant AAV capsid polypeptide, a subject AAV virion or virus, whenintroduced into a subject provides for production of the heterologousgene product encoded for a period of time of more than 6 months, e.g.,from about 6 months to 20 years or more, or greater than 1 year, e.g.,from about 6 months to about 1 year, from about 1 year to about 2 years,from about 2 years to about 5 years, from about 5 years to about 10years, from about 10 years to about 15 years, from about 15 years toabout 20 years, or more than 20 years. In some embodiments, theadministration regimen is part of a vaccination regimen.

Multiple doses of a subject AAV virion can be administered to anindividual in need thereof. Where multiple doses are administered over aperiod of time, an active agent is administered once a month to aboutonce a year, from about once a year to once every 2 years, from aboutonce every 2 years to once every 5 years, or from about once every 5years to about once every 10 years, over a period of time. For example,a subject AAV virion is administered over a period of from about 3months to about 2 years, from about 2 years to about 5 years, from about5 years to about 10 years, from about 10 years to about 20 years, ormore than 20 years. The actual frequency of administration, and theactual duration of treatment, depends on various factors. In someembodiments, the administration regimen is part of a vaccinationregimen.

The dose to achieve a therapeutic effect, e.g., the dose in vectorgenomes/per kilogram of body weight (vg/kg), will vary based on severalfactors including, but not limited to: route of administration, thelevel of heterologous polynucleotide expression required to achieve atherapeutic effect, the specific disease treated, any host immuneresponse to the viral vector, a host immune response to the heterologouspolynucleotide or expression product (protein), and the stability of theprotein expressed. One skilled in the art can readily determine a viriondose range to treat a patient having a particular disease or disorderbased on the aforementioned factors, as well as other factors.Generally, doses will range from at least about, or more, for example,about 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³ or 1×10¹⁴, or more, vectorgenomes per kilogram (vg/kg) of the weight of the subject, to achieve atherapeutic effect. In some embodiments, treatment is administered at adosage of 5×10¹⁰ vg/kg. In some embodiments, the variant AAVpolypeptides of the present invention can be employed to reduce theamount of total AAV vector or other therapeutic molecule administered toa subject, wherein less total AAV vector or other therapeutic moleculeis administered to a subject when said AAV vector or other therapeuticmolecule is transduced using a variant capsid polypeptide as compared tothe amount of AAV vector or other therapeutic molecule administered to asubject when the AAV vector or other therapeutic molecule is transducedusing a non-variant parent capsid polypeptide in order to obtain asimilar therapeutic effect (i.e., both dosages induce similartherapeutic effects or indistinguishable therapeutic effects). In someembodiments, the total vector or other therapeutic molecule administeredto a subject is reduced by about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80% or more when an AAVvector or other therapeutic molecule is transduced using a variantcapsid polypeptide as compared to when an AAV vector or othertherapeutic molecule is transduced using a non-variant parent capsidpolypeptide in order to obtain a similar therapeutic effect (i.e., bothdosages induce similar therapeutic effects or indistinguishabletherapeutic effects). In some embodiments, the total AAV vector or othertherapeutic molecule administered to a subject is reduced by about 5% toabout 80%, about 10% to about 75%, about 15% to about 65%, about 20% toabout 60%, or about 10% to about 50% when the AAV vector or othertherapeutic molecule is transduced using a variant capsid polypeptide ascompared to when the AAV vector or other therapeutic molecule istransduced using a non-variant parent capsid polypeptide in order toobtain a similar therapeutic effect (i.e., both dosages induce similartherapeutic effects or indistinguishable therapeutic effects).

An effective amount or a sufficient amount can, but need not be,provided in a single administration, may require multipleadministrations, and, can but need not be, administered alone or incombination with another composition (e.g., agent), treatment, protocolor therapeutic regimen. For example, the amount may be proportionallyincreased as indicated by the need of the subject, type, status andseverity of the disease treated or side effects (if any) of treatment.In addition, an effective amount or a sufficient amount need not beeffective or sufficient if given in single or multiple doses without asecond composition (e.g., another drug or agent), treatment, protocol ortherapeutic regimen, since additional doses, amounts or duration aboveand beyond such doses, or additional compositions (e.g., drugs oragents), treatments, protocols or therapeutic regimens may be includedin order to be considered effective or sufficient in a given subject.Amounts considered effective also include amounts that result in areduction of the use of another treatment, therapeutic regimen orprotocol, such as administration of recombinant clotting factor proteinfor treatment of a clotting disorder (e.g., hemophilia A or B).

An effective amount or a sufficient amount need not be effective in eachand every subject treated, or a majority of treated subjects in a givengroup or population. An effective amount or a sufficient amount meanseffectiveness or sufficiency in a particular subject, not a group or thegeneral population. As is typical for such methods, some subjects willexhibit a greater response, or less or no response to a given treatmentmethod or use. Thus, appropriate amounts will depend upon the conditiontreated, the therapeutic effect desired, as well as the individualsubject (e.g., the bioavailability within the subject, gender, age,etc.).

With regard to a disease or symptom thereof, or an underlying cellularresponse, a detectable or measurable improvement includes a subjectiveor objective decrease, reduction, inhibition, suppression, limit orcontrol in the occurrence, frequency, severity, progression, or durationof the disease, or complication caused by or associated with thedisease, or an improvement in a symptom or an underlying cause or aconsequence of the disease, or a reversal of the disease.

Thus, a successful treatment outcome can lead to a “therapeutic effect,”or “benefit” of decreasing, reducing, inhibiting, suppressing, limiting,controlling or preventing the occurrence, frequency, severity,progression, or duration of a disease, or one or more adverse symptomsor underlying causes or consequences of the disease in a subject.Treatment methods and uses affecting one or more underlying causes ofthe disease or adverse symptoms are therefore considered to bebeneficial. A decrease or reduction in worsening, such as stabilizingthe disease, or an adverse symptom thereof, is also a successfultreatment outcome.

A therapeutic benefit or improvement therefore need not be completeablation of the disease, or any one, most or all adverse symptoms,complications, consequences or underlying causes associated with thedisease. Thus, a satisfactory endpoint is achieved when there is anincremental improvement in a subject's disease, or a partial decrease,reduction, inhibition, suppression, limit, control or prevention in theoccurrence, frequency, severity, progression, or duration, or inhibitionor reversal, of the disease (e.g., stabilizing one or more symptoms orcomplications), over a short or long duration of time (hours, days,weeks, months, etc.). Effectiveness of a method or use, such as atreatment that provides a potential therapeutic benefit or improvementof a disease, can be ascertained by various methods.

Disclosed methods and uses can be combined with any compound, agent,drug, treatment or other therapeutic regimen or protocol having adesired therapeutic, beneficial, additive, synergistic or complementaryactivity or effect. Exemplary combination compositions and treatmentsinclude second actives, such as, biologics (proteins), agents and drugs.Such biologics (proteins), agents, drugs, treatments and therapies canbe administered or performed prior to, substantially contemporaneouslywith or following any other method or use of the invention, for example,a therapeutic method of treating a subject for a liver disease disease.

The compound, agent, drug, treatment or other therapeutic regimen orprotocol can be administered as a combination composition, oradministered separately, such as concurrently or in series orsequentially (prior to or following) delivery or administration of anAAV vector or AAV virion as described herein. The invention thereforeprovides combinations where a method or use of the invention is in acombination with any compound, agent, drug, therapeutic regimen,treatment protocol, process, remedy or composition, set forth herein orknown to one of skill in the art. The compound, agent, drug, therapeuticregimen, treatment protocol, process, remedy or composition can beadministered or performed prior to, substantially contemporaneously withor following administration of an AAV vector or AAV virion as describedherein, to a subject. Specific non-limiting examples of combinationembodiments therefore include the foregoing or other compound, agent,drug, therapeutic regimen, treatment protocol, process, remedy orcomposition.

Methods and uses of the invention also include, among other things,methods and uses that result in a reduced need or use of anothercompound, agent, drug, therapeutic regimen, treatment protocol, process,or remedy. For example, for a blood clotting disease wherein a patienthas defective blood clotting secretion from the liver, a method or useof the invention has a therapeutic benefit if, in a given subject, aless frequent or reduced dose or elimination of administration of arecombinant clotting factor protein to supplement for the deficient ordefective (abnormal or mutant) endogenous clotting factor secretion fromthe subject liver. Thus, in accordance with the invention, methods anduses of reducing need or use of another treatment or therapy areprovided.

The invention is useful in animals including veterinary medicalapplications. Suitable subjects therefore include mammals, such ashumans, as well as non-human animals. In some embodiments, the human ismale. In some embodiments, the human is female. The term “subject”refers to an animal, typically a mammal, such as humans, non-humanprimates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), adomestic animal (dogs and cats), a farm animal (poultry such as chickensand ducks, horses, cows, goats, sheep, pigs), experimental animals(mouse, rat, rabbit, guinea pig), as well as other avian species (birdsinclude but are not limited to parrots, parakeets (small and large),penguins, cockatiels, lovebirds, parrotlets, caiques, conures, lories,lorikeets, pionus parrots, Poicephalus, canaries, finches, cockatoos,macaws, crows, doves, pigeons, mynah birds, and toucans). Human subjectsinclude fetal, neonatal, infant, juvenile and adult subjects. Subjectsinclude animal disease models, for example, mouse and other animalmodels of blood clotting diseases and others known to those of skill inthe art.

Non-limiting particular examples of liver diseases and disorders includebut are not limited to any conditions that stop the liver fromfunctioning properly or prevent it from functioning well (i.e.,functioning at normal levels). Symptoms of liver diseases and disorderscan include but are not limited to abdominal pain, yellowing of the skinor eyes (jaundice), abnormal results of liver function tests, liverfattening (including, for example, disproportional fattening), andcirrhosis of the liver. Liver diseases and disorders further include butare not limited to: amebic liver abscess, autoimmune liver diseases anddisorders (including, for example, autoimmune hepatitis, primary biliarycirrhosis (PBC), and primary sclerosing cholangitis (PSC)), biliaryatresia, cirrhosis, coccidioidomycosis, delta agent (hepatitis D),drug-induced cholestasis, hemochromatosis, viral hepatitis (including,for example, Hepatitis A, Hepatitis B, and Hepatitis C), neonatalhepatitis, hepatocellular carcinoma (HCC, as well as other livercancer), fibrolamellar hepatocellular carcinoma, liver disease due toalcohol, pyogenic liver abscess, Reye's syndrome, Sclerosingcholangitis, Wilson's disease, acute liver failure (caused by, forexample, drugs, toxins, and various other diseases), alcoholic liverdisease, autoimmune-associated diseases, Budd-Chiari syndrome,hypercoagulable disorders, parasitic infection, chronic bile ductobstruction (including, for example, due to tumors, gallstones,inflammation, and trauma), hemochromatosis, alpha-1 antitrypsin (A1A)deficiency, Wilson disease, Alagille Syndrome, cystic disease of theliver, galactosemia, Gilbert's Syndrome, hemochromatosis, liver diseasein pregnancy, Lysosomal Acid Lipase Deficiency (LALD), porphyria,sarcoidosis, Type 1 Glycogen Storage Disease, Tyrosinemia, Alveolarhydatid disease, bacillary peliosis, congenital hepatic fibrosis,congestive hepatopathy, gastric antral vascular ectasia, hepaticencephalopathy, hepatolithiasis, hepatopulmonary syndrome, hepatorenalsyndrome, hepatosplenomegaly, hepatotoxicity, Indian childhoodcirrhosis, Laennec's cirrhosis, Lyngstadaas Syndrome, peliosis hepatis,progressive familial intrahepatic cholestasis, Zahn infarct, Zieve'ssyndrome, and nonalcoholic fatty liver disease (NAFLD).

Non-limiting particular examples of diseases treatable in accordancewith the invention include those set forth herein as well as a lungdisease (e.g., cystic fibrosis), a blood coagulation or bleedingdisorder (e.g., hemophilia A or hemophilia B with or withoutinhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), epilepsy, lysosomal storage diseases, a copper or ironaccumulation disorders (e.g., Wilson's or Menkes disease) lysosomal acidlipase deficiency, a neurological or neurodegenerative disorder, cancer,type 1 or type 2 diabetes, Gaucher's disease, Hurler's disease,adenosine deaminase deficiency, a metabolic defect (e.g., glycogenstorage diseases), a retinal degenerative disease (such as RPE65deficiency or defect, choroideremia, and other diseases of the eye), anda disease of a solid organ (e.g., brain, liver, kidney, heart), as wellas muscle diseases including not limited to Acid Maltase Deficiency(AMD), Amyotrophic Lateral Sclerosis (ALS), Andersen-Tawil Syndrome,Becker Muscular Dystrophy (BMD), Becker Myotonia Congenita, BethlemMyopathy, Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy),Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency (CPTDeficiency), Central Core Disease (CCD), Centronuclear Myopathy,Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD),Congenital Myasthenic Syndromes (CMS), Congenital Myotonic Dystrophy,Cori Disease (Debrancher Enzyme Deficiency), Debrancher EnzymeDeficiency, Dejerine-Sottas Disease (DSD), Dermatomyositis (DM), DistalMuscular Dystrophy (DD), Duchenne Muscular Dystrophy (DMD), DystrophiaMyotonica (Myotonic Muscular Dystrophy), Emery-Dreifuss MuscularDystrophy (EDMD), Endocrine Myopathies, Eulenberg Disease (ParamyotoniaCongenita), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD),Finnish (Tibial) Distal Myopathy, Forbes Disease (Debrancher EnzymeDeficiency), Friedreich's Ataxia (FA), Fukuyama Congenital MuscularDystrophy, Glycogenosis Type 10, Glycogenosis Type 11, Glycogenosis Type2, Glycogenosis Type 3, Glycogenosis Type 5, Glycogenosis Type 7,Glycogenosis Type 9, Gowers-Laing Distal Myopathy, Hauptmann-ThanheuserMD (Emery-Dreifuss Muscular Dystrophy), Hereditary Inclusion-BodyMyositis, Hereditary Motor and Sensory Neuropathy (Charcot-Marie-ToothDisease), Hyperthyroid Myopathy, Hypothyroid Myopathy, Inclusion-BodyMyositis (IBM), Inherited Myopathies, Integrin-Deficient CongenitalMuscular Dystrophy, Kennedy Disease (Spinal-Bulbar Muscular Atrophy),Kugelberg-Welander Disease (Spinal Muscular Atrophy), LactateDehydrogenase Deficiency, Lambert-Eaton Myasthenic Syndrome (LEMS),Limb-Girdle Muscular Dystrophy (LGMD), Lou Gehrig's Disease (AmyotrophicLateral Sclerosis), McArdle Disease (Phosphorylase Deficiency),Merosin-Deficient Congenital Muscular Dystrophy, Metabolic Diseases ofMuscle, Mitochondrial Myopathy, Miyoshi Distal Myopathy, Motor NeuroneDisease, Muscle-Eye-Brain Disease, Myasthenia Gravis (MG), MyoadenylateDeaminase Deficiency, Myofibrillar Myopathy, MyophosphorylaseDeficiency, Myotonia Congenita (MC), Myotonic Muscular Dystrophy (MMD),Myotubular Myopathy (MTM or MM), Nemaline Myopathy, Nonaka DistalMyopathy, Oculopharyngeal Muscular Dystrophy (OPMD), ParamyotoniaCongenita, Pearson Syndrome, Periodic Paralysis, Peroneal MuscularAtrophy (Charcot-Marie-Tooth Disease), Phosphofructokinase Deficiency,Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency,Phosphorylase Deficiency, Phosphorylase Deficiency, Polymyositis (PM),Pompe Disease (Acid Maltase Deficiency), Progressive ExternalOphthalmoplegia (PEO), Rod Body Disease (Nemaline Myopathy), SpinalMuscular Atrophy (SMA), Spinal-Bulbar Muscular Atrophy (SBMA), SteinertDisease (Myotonic Muscular Dystrophy), Tarui Disease(Phosphofructokinase Deficiency), Thomsen Disease (Myotonia Congenita),Ullrich Congenital Muscular Dystrophy, Walker-Warburg Syndrome(Congenital Muscular Dystrophy), Welander Distal Myopathy,Werdnig-Hoffmann Disease (Spinal Muscular Atrophy), and ZASP-RelatedMyopathy.

Ocular diseases that can be treated or prevented using a subject methodinclude, but are not limited to, selected from acute macularneuroretinopathy; macular telangiectasia; Behcet's disease; choroidalneovascularization; diabetic uveitis; histoplasmosis; maculardegeneration, such as acute macular degeneration, Scorsby's maculardystrophy, early or intermediate (dry) macular degeneration, or a formof advanced macular degeneration, such as exudative macular degenerationor geographic atrophy; edema, such as macular edema, cystoid macularedema and diabetic macular edema; multifocal choroiditis; ocular traumaaffecting a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative and non-proliferative diabetic retinopathy),proliferative vitreoretinopathy (PVR), retinal arterial occlusivedisease, retinal detachment, uveitic retinal disease; sympatheticopthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; aposterior ocular condition caused by or influenced by an ocular lasertreatment; posterior ocular conditions caused by or influenced by aphotodynamic therapy, photocoagulation, radiation retinopathy;epiretinal membrane disorders; central or branch retinal vein occlusion;anterior ischemic optic neuropathy, non-retinopathy diabetic retinaldysfunction; retinitis pigmentosa; retinoschisis; and glaucoma.

In one embodiment, a method or use of the invention includes: (a)providing an AAV virion whose capsid comprises the variant AAV capsidpolypeptides prepared as described herein, wherein the AAV virioncomprises a heterologous nucleic acid sequence, wherein the heterologousnucleic acid sequence is operably linked to an expression controlelement conferring transcription of said nucleic acid sequence; and (b)administering an amount of the AAV virion to the subject such that saidheterologous nucleic acid is expressed in the subject.

In one embodiment, a method or use of the invention includes: (a)providing a therapeutic molecule (including, for example, a vaccine)packaged by variant AAV capsid polypeptides prepared as describedherein, wherein the therapeutic molecule comprises a heterologousnucleic acid sequence, wherein the heterologous nucleic acid sequence isoperably linked to an expression control element conferringtranscription of said nucleic acid sequence; and (b) administering anamount of the therapeutic molecule (including, for example, a vaccine)packaged by variant AAV capsid polypeptides to the mammal such that saidheterologous nucleic acid is expressed in the mammal.

In another embodiment, a method or use of the invention includesdelivering or transferring a heterologous polynucleotide sequence into amammal or a cell of a mammal, by administering a heterologouspolynucleotide packaged by a variant AAV capsid polypeptide, a pluralityof heterologous polynucleotides packaged by variant AAV capsidpolypeptides, an AAV virion prepared as described herein, or a pluralityof AAV virions comprising the heterologous nucleic acid sequence to amammal or a cell of a mammal, thereby delivering or transferring theheterologous polynucleotide sequence into the mammal or cell of themammal. In some embodiments, the heterologous nucleic acid sequenceencodes a protein expressed in the mammal, or where the heterologousnucleic acid sequence encodes an inhibitory sequence or protein thatreduces expression of an endogenous protein in the mammal.

By way of example, respecting hemophilia, it is believed that, in orderto achieve a therapeutic effect, the liver must secrete a level ofcirculating blood coagulation factor to a concentration that is greaterthan 1% of factor concentration found in a normal individual is neededto change a severe disease phenotype to a moderate one. A severephenotype is characterized by joint damage and life-threatening bleeds.To convert a moderate disease phenotype into a mild one, it is believedthat a circulating blood coagulation factor concentration greater thanabout 5% of normal is needed. With respect to treating such a hemophilicsubject, a typical dose is at least 1×10¹⁰ AAV vector genomes (vg) perkilogram (vg/kg) of the weight of the subject, or between about 1×10¹⁰to about 1×10¹¹ vg/kg of the weight of the subject, or between about1×10¹¹ to about 1×10¹² vg/kg of the weight of the subject, or betweenabout 1×10¹² to about 1×10¹³ vg/kg of the weight of the subject, toachieve a desired therapeutic effect. In some embodiments, treatment isadministered at a dosage of 5×10¹⁰ vg/kg.

EXAMPLES Example 1: Novel Recombinant Adeno-Associated Virus CapsidsResistant to Pre-Existing Human Neutralizing Antibodies

Purpose:

To evolve new recombinant AAV (rAAV) capsids which have the combinedability to both transduce human cells, but also the ability to evadeneutralization by pre-existing anti-capsid antibodies in patients.Creation and identification of rAAV vectors that are resilient toneutralization by pre-existing antibody titers would have great utilityin human gene therapy as these vectors are used in clinical trials fornucleic acid delivery. There is a high percentage of the humanpopulation (varies between 25 to 75%) who have pre-existing anti-AAVantibodies that would preclude successful gene transfer for thetreatment of the intended disease. As such, pre-screened patients oftenhave to be excluded from clinical trials and not receive the therapeutictreatment.

Technical Description (Abstract):

Wild-type replicating AAV libraries of 10e5 (aka, 105) variants via DNAshuffling of ten different parental AAV capsids (AAVs 1, 2, 3b, 4, 5, 6,8, 9_hu14, avian, bovine) were utilized. The AAV capsid librariesselectively replicate in human cells when co-administered with wild-typeadenovirus type 5, making chimeric humanized liver mice an excellenttool to allow for selection of capsids with tropism for human liver.Screens were carried out for five rounds of selection inxenotransplanted humanized liver mice and rather than waiting for thescreen to go to completion, the screen ended with some library diversityremaining for use in a subsequent screen for neutralizing antibodyevasion. All variants from round five of the human liver screen werecarried forward and screened for two additional rounds for their abilityto resist binding to pooled human immunoglobulins in IgG immunocaptureassays. The top 100 highly selected variants were sequenced andvectorized into CAG promoter-driven GFP preparations in seven poolsbased on capsid relatedness at the amino acid level. Each pool was thenresubjected to additional pooled human immunoglobulin screening and thebest pool was chosen. The top seven candidates from this pool weretested alongside control serotypes that represent the extremes forhumoral neutralization: highest neutralization (AAV-2), lowestneutralization (AAV-DJ), as well as LK03 that falls in between. The mostrelevant variants from these pools for clinical use were identified tobe AAV-NP84 (SEQ ID NO:2), AAV-NP40 (SEQ ID NO:6) and AAV-NP59 (SEQ IDNO:4). AAV-NP84 (SEQ ID NO:2) has neutralization profiles on par withAAV-DJ and subsequent transduction tests in humanized liver mice in vivodemonstrated a high tropism and specificity for human hepatocytes withAAV-NP84 (SEQ ID NO:2). In comparison, AAV-NP40 (SEQ ID NO:6) has modestneutralization profiles but also shows high human hepatocyte tropism andspecificity in vivo. Finally, AAV-NP59 (SEQ ID NO:4) also displays amodest neutralization profile but is capable of transducing both humanand mouse hepatocytes. This could be very useful as AAV-NP59 (SEQ IDNO:4) could be used to model both pre-clinical studies in mouse as wellas used clinically in humans due to its combined transductioncapabilities.

Such Variant AAV Capsid Polypeptides Find Use in Human Gene Therapy.

AAV Vectors:

Current AAV capsids with excellent tropism to liver hepatocytes include:AAV-8, AAV-DJ, and AAV-LK03. However, both AAV-8 and AAV-DJ largely onlytransduce mouse heptocytes, not human hepatocytes. Although AAV-LK03transduces human hepatocytes well in humanized mice in vivo, about ⅓ ofpotential patients have pre-existing antibodies against it, limiting itsability to be useful in these patients. The new AAV capsids AAV-NP84(SEQ ID NO:2), AAV-NP40 (SEQ ID NO:6) and AAV-NP59 (SEQ ID NO:4) showrobust human hepatocyte transduction in humanized mice in vivo. AAV-NP84(SEQ ID NO:2) also shows what appears to be a favorable antibodyneutralization profile.

Variations in capsid sequence might enhance the neutralization profileeven further, enhance transduction of human tissues and/or expandtransduction of various human cell types that are good targets fortherapeutic interventions.

Capsid sequences AAV-NP84 (SEQ ID NO:2), AAV-NP40 (SEQ ID NO:6) andAAV-NP59 (SEQ ID NO; 4), as well as AAV-NP30 (SEQ ID NO:8) have beenidentified in the present invention.

Novel variant AAV capsids that exhibit both human hepatocyte tropism andhuman immune evasion abilities are provided in the present invention.

Example 2: Bioengineered AAV Capsids with Combined High Human LiverTransduction In Vivo and Unique Humoral Seroreactivity

Abstract:

Existing recombinant adeno-associated virus (rAAV) serotypes fordelivering in vivo gene therapy treatments for human liver diseases havenot yielded combined high-level human hepatocyte transduction andfavorable humoral neutralization properties. Yet, these combinedproperties are important for therapeutic efficacy. To bioengineercapsids that exhibit both unique seroreactivity profiles andfunctionally transduce human hepatocytes at therapeutically relevantlevels, multiplexed sequential directed evolution screens were performedusing diverse capsid libraries in both primary human hepatocytes in vivoand with pooled human sera from thousands of patients. AAV librarieswere subjected to five rounds of in vivo selection in mice withhumanized livers to isolate an enriched human-hepatotropic library thatwas then used as input for a sequential on-bead screen against pooledhuman immunoglobulins. Evolved variants were vectorized and validatedagainst existing hepatotropic serotypes. Two of the evolved AAVserotypes—NP40 and NP59—exhibited both improved functional humanhepatocyte transduction in vivo in chimeric humanized liver mice, alongwith favorable human seroreactivity profiles. These novel capsidsrepresent enhanced vector delivery systems for future human liver genetherapy applications.

Introduction:

Despite decades of liver gene therapy research, no liver disordertreated with rAAV gene transfer to date has reached curative, ratherthan therapeutic, levels. Although there has been substantial progressin treating patients with rAAV vectors expressing human Factor IX inhemophilia B^(1,2), transduction was low but compensated for withsupraphysiologic levels of expression from the few transducedhepatocytes^(3,4). For non-cell autonomous diseases like hemophilia Bthat have the benefit of secreted elements, these low transductionlevels may be sufficient when paired with transfer vectors optimized forhigh expression. However, such transduction levels will be suboptimalfor liver diseases with more demanding cell autonomous phenotypes.Numerous hurdles remain for improving long-term functional human livertransduction including increasing total functional hepatocytetransduction levels, pre-existing neutralizing antibodies (nAbs) againstrAAV capsids, and cellular immune responses to capsid peptides presentedon transduced hepatocytes. Suboptimal transduction to date likely stemsfrom the fact that preclinical rAAV selection and validation hashistorically been performed in animal models that neither recapitulatethe hepatocellular tropism in humans, nor the kinetics and strength ofexpression which can be reached^(1,5). Humoral neutralization of rAAV inthe bloodstream arises from patient exposure to parental serotypes innature⁶⁻¹¹. Immune-mediated destruction of transduced hepatocytes is dueto CD8+ T-cell responses to rAAV capsid components¹², but this canlargely be managed via corticosteroid administration¹ and reduceddosing. Thus, high-level, functional human hepatocyte transduction andevading humoral neutralization remain the leading barriers to trulyefficacious clinical liver gene therapy today.

Importantly, rAAV vectors can be bioengineered to achieve transductionand neutralization potentials not possible with parental serotypesthrough directed evolution of diverse capsid libraries¹³⁻¹⁵. Thistechnique utilizes replicating AAV throughout the entire selection andevolution process. In contrast to non-replicating screens which onlyselect for receptor binding and uptake^(16,17), approaches that usereplicating AAV select for every step in the intrahepatocellulartrafficking and expression cascade, all of which can heavily influencethe efficiency of transduction post-entry^(5,18-20.) Even single aminoacid capsid mutations have been shown to affect functional transductionpost-entry and post-uncoating²¹. Taken together, these data support theuse of replicating AAV screens whenever possible.

Here each of these important parameters were combined: utilizingreplicating AAV libraries that allow for selection beyond justhepatocyte receptor binding and entry, evolving human hepatocyte tropismin human rather than mouse hepatocytes in vivo, screening for humoralevasion against pools of human immunoglobulins from thousands ofpatients, and assessing transduction using clinically meaningfulmethodologies. The result is a panel of novel rAAV variants withsuperior human hepatic transduction and unique humoral neutralizationcompared to previously characterized serotypes.

Results:

No Existing rAAV Serotype Fulfills all the Necessary Criteria for IdealLiver Delivery in Human

There exists in the gene therapy field no ideal rAAV vector candidatethat satisfies the combinatorial needs of high functional humanhepatocyte transduction and low neutralization potential. While severalcandidates have demonstrated detectable human hepatocyte transduction ineither clinical trials or in xenograft models, none exhibit favorableneutralization profiles and vice versa (see Table 3, below). Any rAAVthat has not been tested in either a human liver trial or in humanizedliver mice was excluded from this list, as assessing human hepatocytetransduction solely in cell lines (rAAV-F series²²) or non-humanizedmice (rAAV1^(23,24)) has been shown to have poor correlation with humanliver transduction^(1,5). The two candidates with the most favorableextremes are rAAV-LK03 which has high human hepatocyte transduction⁴ butalso elicits substantial nAb levels⁹, and rAAV-DJ which elicits low nAblevels¹³ but has yet to be assessed for human hepatocyte transduction.To address this, six humanized FRG²⁵ mice were treated withssAAV-DJ-CAG-GFP and measured transduction in mouse and humanhepatocytes. Liver immunohistochemistry for human FAH and viral GFPdemonstrated low levels of functional human hepatocyte transduction(<5%) in all treated mice (see FIG. 17a, b ). The results from GFP RNAFISH followed by sequential GFP DNA FISH on treated liver sectionsdemonstrated that the block to functional human transduction occurredpost-uncoating (see FIG. 17c-e ). Although both strong nuclear GFP DNAas well as nuclear and cytoplasmic GFP RNA were detected, there was noGFP protein, indicating a translational block to functional expression.

TABLE 3 Literature Comparison Estimated human hepatocyte Humanhepatocyte AAV transduction in transduction in Mouse hepatocyte Levelsof pre-existing nAb in Serotype clinical trials humanized micetransduction humans AAV2 Low² Low⁴ Low^(4,18,52)Medium^(27,35)-high^(9,10,27,28,34) AAV3b ND Medium³ Low^(3,24,53)High^(9,28) AAV5 Low^(54,55)* Low³ Low^(3,18,24,56)Low^(35,55)*-medium^(10,28,34,35)-high²⁸ AAV8 Low^(1,5) Low^(3,4)Medium³-high^(4,57) Medium^(27,34)-high^(9,10,28) AAV9 ND Low³ Medium³Medium³⁴ AAV-LK03 ND High⁴ Low⁴ High⁹ AAV-DJ ND Low (see FIG. S1)High^(13,58) Low¹³ (and see FIG. 5)

Table 3 provides a literature comparison of functional hepatocytetransduction and pre-existing neutralizing antibody levels in humanswith existing hepatotropic AAV serotypes. The ability to functionallytransduce hepatocytes in vivo in different settings was compared. Column1 shows estimated functional percent human hepatocyte transduction fromclinical trials. All values are estimates since no biopsies assessingfunctional transduction (protein expression rather than vector copynumber) post-treatment have been performed. Of note, while expressionlevels from AAV8 were therapeutic in some patients (sustained 5-7% ofnormal human FIX levels), previous data showing supraphysiologicexpression per transduced hepatocyte suggests <10% of patienthepatocytes were transduced. Column 2 shows actual percent humanhepatocyte functional transduction in vivo measured from treatedxenografted liver mice. Column 3 shows actual percent mouse hepatocytefunctional transduction in vivo from non-xenografted mice of varyinggenotypes and strain backgrounds. Column 4 shows levels of pre-existingneutralizing antibodies (nAb) measured from human sera in neutralizationassays. ND=not determined. *=predicted (data acquired from anon-peer-reviewed press release prior to the end of the study). For thethree transduction columns, low=0-10%, medium=10-50%, high=50-100% ofliver hepatocytes. For the neutralization column, low=0-10%,medium=10-50%, high=50-100% of patients whose sera showed neutralizationfor that AAV capsid.

Diverse AAV Capsid Library Screening in Primary Human Hepatic XenograftsIn Vivo

The ideal rAAV capsid for liver-directed gene therapy in humans wouldcombine high functional hepatic transduction with immune evasion. Toevolve such a variant, a directed evolution approach that bioengineereddiverse capsids through DNA shuffling of capsid genes from numerousgenetically and functionally diverse parental AAV serotypes wasemployed. Enzymatic fragmentation followed by assembly of shuffledfull-length capsid genes was used to generate a diverse capsid library.Those were then cloned into an AAV shuttle vector and utilized toproduce live replicating AAV libraries (see FIG. 2a , 18). The librarywas produced from 10 different parental capsid serotypes: 1, 2, 3b, 4,5, 6, 8, 9_hu14, avian and bovine. To maximize the likelihood that theshuffled capsids could both evade humoral neutralization andfunctionally transduce human hepatocytes, multiplexed sequential screensusing primary human tissues were performed. First, primary humansurgical liver specimens were digested and purified to isolatepopulations of human hepatocytes for transplantation into FRG mice (seeFIG. 2b ). 5E10 vector genomes (vg) of AAV library was administeredintravenously into xenografted mice (see FIG. 2c ) followed by liveAdenovirus-5 (Ad-5) 24-hrs later. Replicated AAV variants thattrafficked successfully to the liver were isolated after 2 days post-Ad5administration, minimally purified and re-titered, and again injected at5E10 vg/mouse into new xenotransplanted mice. This in vivo screeningcycle was carried out for five rounds of selection. Diversity monitoringvia Sanger sequencing began at round 3, and each round thereafter, untilround 5 when sufficient pressure for human hepatic transduction had beenselected but some library diversity remained. This enrichedhuman-hepatotropic AAV library was then used as input for a series ofsequential on-bead screens against pooled human immunoglobulins fromthousands of patients to select variants with reduced humoralneutralization potential across the general population (see FIG. 2d ).After two rounds of binding selection, those variants that remainedunbound were subjected to stringent characterization for potentialclinical utility.

Identifying Functionally Important Residues Via Structural andComparative Computational Modeling

At the completion of the second sequential screen, capsid sequencesamplified from the input library and several selection rounds were deepsequenced using PacBio single-molecule sequencing. Round-to-roundpositional analyses from thousands of capsids identified the selectionfor key residues (see FIG. 12). This approach was more revealing thanclassic phylogenetic trees that root on the nearest full-length parentalsequence, effectively masking functionally important residues withinfull-length capsid relatedness (see FIG. 19). Interestingly, althoughrAAV2 is known to be a poor functional transducer of human hepatocytes,several structural fragments from AAV2 were highly selected in theinitial screen during the early rounds of screening, most notablyresidues in the C-terminal end of VP3. However, many stretches of AAV2sequence were strongly selected against including: a portion of theunique region of VP1 and the unique region of VP2 (aligned residues67-to-146), which instead selected for AAV8 residues; a large stretch ofVP3 (aligned residues 321-to-423) which selected for AAV3b residues; andseveral high frequency de novo mutation hot spots (aligned residues 42,158, 165, 181, 290, 515 and 555) which contained various amino acids notpresent in any of the parental serotypes used for library generation. Asthe experiments transitioned into the second screen to select capsidvariants capable of humoral evasion, the IgG-bound and unbound AAVvariants exhibited a high degree of structural mean similarity. Only afew key regions were different between them that are likely necessary incombination to achieve the improved IgG evasion. Here, the globaldifferences were more easily seen with the individual, rather thanaggregate, full-length capsid sequences from PacBio single-moleculesequencing (see FIG. 19a ).

To further probe the evolved variants, several of the most highlyselected capsid variants obtained after the final screen were chosen(see FIG. 20a ) and vectorized with GFP or Firefly Luciferase (FLuc)expression constructs for subsequent validation experiments. Todetermine the genetic contribution of each parental AAV serotype to theevolved capsids fragment crossover mapping (see FIG. 13a ), structuralcapsid mapping (see FIG. 13b ) and predictive fragment conservationanalyses (see FIG. 20b ) were performed. These complementarymethodologies demonstrated selection for certain residues andhighlighted both unique and shared domains. Shuffling was achieved alongthe length of Cap, including VP1, VP2, VP3 and AAP. The parentalserotypes that contributed the most to the evolved variants includedAAV2, 3b, 1 and 6 in that order. None of the selected variants hadappreciable contributions from AAV4, 5, 8, 9_hu14, bovine or avian. Itis interesting to note the lack of selection for almost any unique AAV8sequence in variants selected for their ability to transduce human asopposed to mouse hepatocytes. This supports previous findings of some ofthe inventors⁴ and that of others in the field³ that rAAV8 is a poorfunctional transducer of human hepatocytes in vivo, and is much bettersuited for mouse transduction studies.

Shuffled NP40, NP59 and NP84 capsid sequences (see FIG. 13a , 23)contained many fragments from parental serotypes with known livertropism, and these variants were selected for in the initial in vivoscreen in humanized liver xenografts. Each of these 3 shuffled capsidswould be predicted to have similar comparative structures to one anothergiven their highly similar capsid sequences (see FIG. 13a, b ). NP40 isthe most shuffled of the three, with the unique region of VP1 fromAAV1/3b/6/8/9, the unique region of VP2 derived from AAV2, and finallyVP3 with contributions from AAV2 and 3b as well as one de novo mutation(K555E). As with the other variant capsids, a conserved contributionfrom AAV3b at positions 326-426 suggesting that this is the minimalstructural region from AAV3b required for enhanced human hepatictransduction was observed. NP59 is similar to NP40 but lacks the diverseVP1 contributions and is instead composed of AAV2 in that sequencestretch. NP59 has the same VP2 and VP3 contributions as NP40 except forone de novo mutation (N622D). NP84 shares the unique regions of VP1 andVP2 with NP59, but has a much larger contribution from AAV3b and lessfrom AAV2 in VP3, as well as two de novo mutations (K555E and R611G).Looking globally at all three variant capsids, structural mappinghighlighted the subtle structural heterogeneity in hypervariable regionsbut also macro-conservation within key structural domains such as thecylinder (from AAV2), canyon (from AAV3b) and various symmetry axes.

Liver Xenografts are an Accurate Surrogate for Assessing Human HepaticTransduction

To rigorously assess the functional human hepatic transductioncapabilities of the shuffled capsids in an appropriate in vivo setting,humanized FRG xenograft mice were transduced. To reduce bias andmaximize stringency, cohorts of xenografted liver mice were produced intwo different labs and administered variant (NP40, NP59 and NP84) orcontrol (LK03 and DJ) rAAV capsids expressing GFP. Humanized mice ateach of the two locations were administered rAAV at the same dose(2E11/mouse), via the same delivery method (intravenous lateral tailvein injection), and assessed for transduction via GFPimmunohistochemistry 14-days post-AAV administration (see FIG. 14a, b ).Although different promoters were used, both CAG and LSP1 have beenshown to express at equivalent levels in hepatocytes²⁶. To assess thepotential impact of repopulation percentage on transduction, one cohortwas transduced at high repopulation levels and the other with lowrepopulation levels. The independent results from two blinded labsdemonstrated that shuffled variants NP40 and NP59 (and in the highrepopulation cohort, also NP84) had significantly increased functionalhuman hepatocyte transduction over control serotypes LK03 and DJ (seeFIG. 14c , 17 a, b). Although the trend for increased transduction byvariants over controls held regardless of the degree of repopulation,the average transduction level varied depending on the availability ofhuman hepatocytes and limiting rAAV virions. Transduction was seenacross the hepatic lobule where gradients in metabolic activity, andpossibly expression and secretion of transgene products exist. The newshuffled variants are highly specific for transduction of humanhepatocytes. When injected IV into non-humanized Balb-CJ mice, theshuffled variants either functionally transduced the liver very poorlyor not at all (see FIG. 21a, b ). This is in sharp contrast to rAAV-DJwhich outperformed even rAAV8 at functional mouse liver transduction.

Immunologic Properties of Evolved Hepatotropic AAV Variant

To predict the likelihood of rAAV neutralization in patients withpre-existing and potentially cross-reacting anti-AAV capsid antibodies,both seroreactivity assays and transduction neutralization assays usingserum from a variety of patient groups and nonhuman primates wasperformed. First, individual human serum samples from 50 healthy U.S.adults of each gender (see Table 4, below) were assessed for theirseroreactivity to the shuffled capsid variants and control serotypes(see FIG. 15a , 22 a, b, Table 5, below). Shuffled variants NP40, NP59,NP84 and DJ all had significantly reduced seroreactivity profilescompared to rAAV8 and LK03 (P<0.001-0.0001), known to have high humanneutralization frequencies^(9,10,27,28). Separately assessingseroreactivity by gender demonstrated a statistically significantdifference in seroreactivity against the different capsids in men andwomen, however sample numbers were low and must be interpreted withcaution (see Table 5). While both men and women showed significantlyimproved seroreactivity to all shuffled variants over rAAV8, only maleshad significant improvements compared to LK03 (n=33). Female patientsdid not demonstrate significant seroreactivity differences between LK03and each of the three new variant capsids (NP40, NP59 and NP84), albeitfrom low patient numbers (n=17).

TABLE 4 Details for normal off-clot human serum samples DOS Age GenderEthnicity Smoke ABO DOS Age Gender Ethnicity Smoke ABO Jan. 29, 2016 34Male Black Yes B+ Jan. 29, 2016 55 Male Black Yes A+ Feb. 8, 2016 23Female Caucasian Yes O+ Jan. 29, 2016 22 Male Black Yes O+ Feb. 8, 201637 Female Black No A+ Jan. 29, 2016 52 Male Black Yes B+ Feb. 8, 2016 21Female Black No O+ Jan. 29, 2016 22 Male Black No A+ Feb. 8, 2016 37Female Caucasian Yes A− Jan. 29, 2016 24 Male Black Yes A+ Feb. 8, 201624 Female Caucasian No A− Feb. 5, 2016 18 Female Black Yes A+ Jan. 29,2016 21 Male Black No O+ Jan. 29, 2016 40 Male Black No B+ Jan. 29, 201623 Male Black Yes O+ Jan. 29, 2016 33 Male Black Yes A+ Jan. 29, 2016 25Female Black Yes O+ Jan. 12, 2016 42 Male Black No B+ Jan. 29, 2016 25Male Black Yes O+ Jan. 29, 2016 22 Male Black No O+ Feb. 5, 2016 27Female Black No A+ Jan. 29, 2016 22 Female Black No A+ Jan. 29, 2016 21Male Black Yes B+ Jan. 29, 2016 41 Male Caucasian Yes A− Feb. 8, 2016 21Female Black Yes O+ Jan. 29, 2016 23 Male Black Yes O+ Jan. 29, 2016 20Male Black Yes O+ Jan. 12, 2016 39 Male Caucasian Yes A− Jan. 12, 201631 Male Black Yes A+ Jan. 29, 2016 18 Male Black No O+ Jan. 29, 2016 29Male Black No B+ Jan. 29, 2016 19 Male Black Yes O+ Jan. 29, 2016 42Male Black Yes A+ Jan. 29, 2016 23 Female Black No O+ Jan. 29, 2016 20Male Black Yes O+ Jan. 29, 2016 18 Female Black No A+ Jan. 29, 2016 39Female Black Yes B+ Jan. 29, 2016 42 Male Black Yes A+ Jan. 29, 2016 30Male Black Yes B+ Jan. 29, 2016 24 Male Black Yes O+ Jan. 29, 2016 22Male Black Yes A+ Jan. 29, 2016 53 Male Black Yes A+ Jan. 29, 2016 35Male Black Yes A+ Jan. 12, 2016 24 Male Black No B+ Jan. 29, 2016 24Male Black No A+ Jan. 29, 2016 59 Female Black Yes B+ Feb. 5, 2016 18Female Caucasian No O+ Jan. 29, 2016 22 Female Black Yes O+ Feb. 5, 201636 Female Black No B+ Jan. 29, 2016 43 Male Black No A+

Table 4 provides details for normal off-clot human serum samples.Details on 50 US serum donors from FIG. 15A including date of sampleblood draw (DOS), age at time of donation, gender, ethnicity, smokerstatus and ABO blood type. All donors were negative for HBV, HCV and HIV(data not shown).

TABLE 5 Human seroreactivity statistics Comparisons Mean Diff. 95.00% CIof diff. Significant Summary P Value STATISTICS FOR FIG. 15A AAV8 vs.LK03 0.508 −0.017 to 1.033 No ns 0.0644 AAV8 vs. DJ 2.085  1.56 to 2.61Yes **** <0.0001 AAV8 vs. NP40 1.702  1.177 to 2.227 Yes **** <0.0001AAV8 vs. NP59 1.56  1.035 to 2.085 Yes **** <0.0001 AAV8 vs. NP84 1.232 0.707 to 1.757 Yes **** <0.0001 LK03 vs. DJ 1.577  1.052 to 2.102 Yes**** <0.0001 LK03 vs. NP40 1.194  0.669 to 1.719 Yes **** <0.0001 LK03vs. NP59 1.052 0.5275 to 1.577 Yes **** <0.0001 LK03 vs. NP84 0.724 0.199 to 1.249 Yes ** 0.0013 DJ vs. NP40 −0.382 −0.908 to 0.143 No ns0.2947 DJ vs. NP59 −0.524 −1.049 to 0.001 No ns 0.0504 DJ vs. NP84−0.853  −1.378 to −0.328 Yes **** <0.0001 NP40 vs. NP59 −0.142 −0.667 to0.383 No ns 0.9712 NP40 vs. NP84 −0.47 −0.995 to 0.055 No ns 0.1083 NP59vs. NP84 −0.328 −0.853 to 0.197 No ns 0.4709 STATISTICS FOR FIG. 22AAAV8 vs. LK03 0.129 −0.528 to 0.787 No ns 0.9930 AAV8 vs. DJ 2.043 1.386 to 2.701 Yes **** <0.0001 AAV8 vs. NP40 1.536  0.879 to 2.193 Yes**** <0.0001 AAV8 vs. NP59 1.461  0.804 to 2.119 Yes **** <0.0001 AAV8vs. NP84 0.915  0.257 to 1.572 Yes ** 0.0013 LK03 vs. DJ 1.914  1.257 to2.572 Yes **** <0.0001 LK03 vs. NP40 1.407  0.749 to 2.064 Yes ****<0.0001 LK03 vs. NP59 1.332 0.675 to 1.99 Yes **** <0.0001 LK03 vs. NP840.786  0.128 to 1.443 Yes ** 0.0092 DJ vs. NP40 −0.508 −1.165 to 0.150No ns 0.2316 DJ vs. NP59 −0.582 −1.239 to 0.075 No ns 0.1151 DJ vs. NP84−1.129  −1.786 to −0.471 Yes **** <0.0001 NP40 vs. NP59 −0.074 −0.732 to0.583 No ns 0.9995 NP40 vs. NP84 −0.621 −1.278 to 0.036 No ns 0.0759NP59 vs. NP84 −0.547 −1.204 to 0.111 No ns 0.1633 STATISTICS FOR FIG.22B AAV8 vs. LK03 1.321  0.606 to 2.037 Yes **** <0.0001 AAV8 vs. DJ2.166   1.45 to 2.881 Yes **** <0.0001 AAV8 vs. NP40 1.967  1.251 to2.682 Yes **** <0.0001 AAV8 vs. NP59 1.638  0.922 to 2.353 Yes ****<0.0001 AAV8 vs. NP84 1.888  1.172 to 2.603 Yes **** <0.0001 LK03 vs. DJ0.845 0.1291 to 1.56  Yes * 0.0113 LK03 vs. NP40 0.646 −0.070 to 1.361No ns 0.1008 LK03 vs. NP59 0.317 −0.399 to 1.032 No ns 0.7885 LK03 vs.NP84 0.567 −0.149 to 1.282 No ns 0.2011 DJ vs. NP40 −0.199  −0.915 to0.5165 No ns 0.9645 DJ vs. NP59 −0.528 −1.244 to 0.188 No ns 0.2706 DJvs. NP84 −0.278 −0.993 to 0.438 No ns 0.8657 NP40 vs. NP59 −0.329 −1.045to 0.387 No ns 0.7605 NP40 vs. NP84 −0.079 −0.794 to 0.637 No ns 0.9995NP59 vs. NP84 0.250 −0.465 to 0.966 No ns 0.9097

Table 5 provides human seroreactivity statistics. Comparativestatistical data for all patients (FIG. 15A); separated male patients(FIG. 22A); and separated female patients (FIG. 22B).

To support future pre-clinical testing in nonhuman primates,seroreactivity with serum from a small cohort of 6 rhesus macaques (seeTable 6, below) against the same panel of AAVs (see FIG. 15b ) wasassessed. Given the small cohort size, no statistically significantdifference was seen for mean seroreactivity between the tested capsids(see Table 7, below) with the exception that seroreactivity against DJwas significantly lower than AAV8 (P<0.01). In vitro neutralizationassays in human 2V6.11 permissive cells using serum from a limitedcohort of 21 healthy human donors from the E.U. found mean similarityacross all serotypes (see FIG. 15c , Table 8, below). Although allshuffled variants had lower mean levels of neutralization than LK03,only rAAV8 reached statistical significance (P<0.001) in this smallcohort (n=21). One important potential application of these capsidsrelates to hemophilia B trials. Thus, seroreactivity assays with serumfrom 21 adult males with hemophilia B (see Table 9, below) wasperformed. Due to sample limitations, the variants to only the leadingcandidate, LK03, was compared. Results showed that compared to LK03,NP59 had more favorable mean seroreactivity in 66% of patients, whileboth NP84 and NP40 were more favorable in 53% of patients, although didnot reach statistical significance in this small cohort (see FIG. 15d ,Table 9). Cumulatively, these findings highlight the uniqueimmunological features of variant capsids NP40, NP59, NP84 and DJ,particularly when it comes to seroprevalence and antibody-mediatedneutralization.

TABLE 6 Nonhuman primate samples Country Age at of ID collection Date ofbirth Gender origin Supplier 2 24 months Oct. 23, 2013 Male MauriceBIOPRIM (France) 3 24 months Oct. 26, 2013 Male Maurice BIOPRIM (France)7 24 months Nov. 8, 2013 Male Maurice BIOPRIM (France) 9 23 months Nov.14, 2013 Male Maurice BIOPRIM (France) 11 23 months Nov. 17, 2013 MaleMaurice BIOPRIM (France) 19 23 months Nov. 13, 2013 Male Maurice BIOPRIM(France)

Table 6 provides details for nonhuman primate serum samples from healthyadult rhesus Macaca fascicularis monkeys. Monkey ID, age at time ofblood draw, date on birth (month/day/year), gender, country of originand supplier are shown.

TABLE 7 Additional statistics from FIG. 15 Comparisons Mean Diff. 95.00%CI of diff. Significant Summary P Value STATISTICS FOR FIG. 15B AAV8 vs.LK03 0.117 −0.141 to 0.375 No ns 0.7285 AAV8 vs. DJ 0.293  0.035 to0.551 Yes * 0.0196 AAV8 vs. NP40 0.228 −0.030 to 0.486 No ns 0.1065 AAV8vs. NP59 0.163 −0.095 to 0.421 No ns 0.4014 AAV8 vs. NP84 0.197 −0.062to 0.455 No ns 0.2132 LK03 vs. DJ 0.176 −0.082 to 0.434 No ns 0.3192LK03 vs. NP40 0.111 −0.1474 to 0.369  No ns 0.7701 LK03 vs. NP59 0.046−0.2125 to 0.304  No ns 0.9936 LK03 vs. NP84 0.080 −0.179 to 0.338 No ns0.9290 DJ vs. NP40 −0.065 −0.323 to 0.193 No ns 0.9688 DJ vs. NP59−0.130 −0.389 to 0.128 No ns 0.6343 DJ vs. NP84 −0.096 −0.355 to 0.162No ns 0.8558 NP40 vs. NP59 −0.065 −0.323 to 0.193 No ns 0.9690 NP40 vs.NP84 −0.031 −0.289 to 0.227 No ns 0.9990 NP59 vs. NP84 0.034 −0.224 to0.292 No ns 0.9984 STATISTICS FOR FIG. 15C AAV8 vs. LK03 −0.725  −1.032to −0.418 Yes **** <0.0001 AAV8 vs. DJ −0.466  −0.773 to −0.159 Yes ***0.0004 AAV8 vs. NP40 −0.422  −0.729 to −0.115 Yes ** 0.0018 AAV8 vs.NP59 −0.567  −0.874 to −0.260 Yes **** <0.0001 AAV8 vs. NP84 −0.433 −0.740 to −0.126 Yes ** 0.0012 LK03 vs. DJ 0.258 −0.049 to 0.565 No ns0.1506 LK03 vs. NP40 0.303 −0.004 to 0.610 No ns 0.0550 LK03 vs. NP590.158 −0.149 to 0.465 No ns 0.6682 LK03 vs. NP84 0.292 −0.015 to 0.599No ns 0.0720 DJ vs. NP40 0.045 −0.262 to 0.352 No ns 0.9982 DJ vs. NP59−0.10 −0.408 to 0.206 No ns 0.9308 DJ vs. NP84 0.034 −0.273 to 0.341 Nons 0.9995 NP40 vs. NP59 −0.146 −0.453 to 0.162 No ns 0.7389 NP40 vs.NP84 −0.011 −0.318 to 0.296 No ns >0.9999 NP59 vs. NP84 0.134 −0.173 to0.441 No ns 0.7983 STATISTICS FOR FIG. 15D NP40 vs. NP59 0.179 −0.093 to0.451 No ns 0.3108 NP40 vs. NP84 0.051 −0.221 to 0.323 No ns 0.9606 NP40vs. LK03 0.041 −0.231 to 0.313 No ns 0.9782 NP59 vs. NP84 −0.129 −0.401to 0.143 No ns 0.5970 NP59 vs. LK03 −0.138 −0.410 to 0.134 No ns 0.5390NP84 vs. LK03 −0.010 −0.282 to 0.262 No ns 0.9997

Table 7 provides additional statistics from FIG. 16. Comparativestatistical data for seroreactivity from 6 adult rhesus macaques (FIG.15B); neutralization from 21 healthy adults from the E.U. (FIG. 15C);and seroreactivity from 21 adult hemophilia B patients (FIG. 15D).

TABLE 8 Human serum samples - healthy donors Country of origin GenderAge 1 France Male 10 2 France Male 3 3 France Male 7 4 France Female 465 France Female 7 6 France Female 22 7 France Female 39 8 France Female30 9 Netherlands Female 28 30 France Male 38 31 France Male 39 32 FranceMale 31 33 France Male 39 34 France Male 63 35 France Female 50 36France Female 39 37 France Female 47 38 France Male 37 39 France Male 6240 France Female 46 41 France Male 53

Table 8 provides details for human serum samples from 21 healthy donorsfrom the E.U. used for the neutralization studies. Subject ID, countryof origin, gender and age (in years) at time of donation are shown.

TABLE 9 Human serum samples - hemophilia B ID Age Gender 1 19 Male 3 45Male 5 19 Male 6 24 Male 7 27 Male 9 37 Male rf206 51 Male rf209 20 Malerf211 30 Male rf212 28 Male rf210 27 Male rf012 25 Male rf016 31 Malerf013 32 Male rf010 22 Male rf011 30 Male rf208 41 Male rf205 53 Malesj008 22 Male rf009 28 Male rf014 66 Male

Table 9 provides details for human serum samples from 21 donors withconfirmed hemophilia B. Patient ID, age at time of donation and genderare shown.

Comparative Human Hepatic Organoid Transduction Assays SupportDifferential Transduction

In addition to treating liver diseases in vivo with intravenous deliveryof rAAV, ex vivo gene delivery or gene correction studies intransplantable human hepatic organoids represents a potential futuretherapy for some liver diseases. To establish whether the evolved livervariants could also be used ex vivo, GFP transduction assessments inprimary human liver organoids were performed. In duplicate organoidtransduction time course assessments spanning 14 days (see FIG. 16a, b), shuffled variant NP40 showed significantly greater functional humanliver organoid transduction over rAAV2 and rAAV8 by mean fluorescentintensity (MFI) imaging of GFP, and had equivalent MFI to that seen fromthe high-level transduction achieved with LK03 and DJ—known strong invitro transducers. NP59 had similar transduction levels as rAAV2 and hadmuch greater MFI than rAAV8. NP84 was not a strong transducer of humanhepatic organoids ex vivo.

Discussion

Choosing the best rAAV serotype for optimum human hepatic delivery hasgrown increasingly complex and controversial in recent years stemmingfrom differences in experimental setup, data interpretation andreproducibility^(3,4,29,30). Given the number of altered variables inexperimental design by different groups, this is perhaps not surprising.No two studies have been performed identically, thus comparisons haveand will continue to be confounded. At its simplest, use of differentmodel systems for assessment has created difficulties. It is well knownthat rAAV transduction in in vitro culture systems does not correlatewith in vivo transduction levels, thus testing in these lines for thepurposes of establishing functional in vivo transduction without othersupportive data should be abandoned. Even with in vivo transductionmeasurements, use of different species with no human xenograft (mice),has been shown to not recapitulate transduction outcomes in humans^(1,5)and such results should be interpreted with caution. While no modelorganism perfectly recapitulates a human liver, current evidencedemonstrates that assessing functional transduction in xenograft livermodels engrafted with primary human hepatocytes has best recapitulatedexisting patient data^(3,4) and should be the collective model movingforward. Similarly, quantifying functional transduction by vector copynumber is grossly inaccurate as it measures all genomes post-entryirrespective of functionality. This is critically importantquantitatively as most AAV genomes do not complete the intracellulartrafficking and expression cascade^(31,32) required for therapeuticrelevance, thus leading to large over-estimations of transduction.

In this study, the potential importance of controlling for and reportingthe repopulation levels in humanized mice since the relative ratio ofmouse to human hepatocytes was likely critical to overall transductionefficiency levels when rAAV virions were limiting was demonstrated. Todate, it has been challenging to even attempt to reproduce studiespublished in the field due to poor methods reporting and low numbers oftreated subjects. To facilitate comparisons between future studies,additional variables should also be stringently controlled and reportedincluding: detailed mouse maintenance conditions, strain backgrounds ofmice or other animal models, human hepatocyte donor characteristics(age, gender, disease state, location), method of injection, AAVproduction and purification methods employed, detailed transfer vectordescriptions (including promoter, transgene, enhancer, polyA, genometype, etc.), method of titration, final resuspension solution, method ofquantifying vector transduction and any normalizations performed.

High levels of preexisting nAbs against parental serotypes in patientstreated to date have made pre-screening a requirement for all futuretrials. Prevalence of nAbs varies with patient geography, health status,gender, age and likely many other variables³³. Among known hepatotropicserotypes, the limited human studies to date have shown highest nAblevels against rAAV2^(9,10,27,28,34), rAAV5^(10,28,34,35),rAAV8^(10,28,27) and rAAV3b^(9,28) along with the highly relatedrAAV-LK03 capsid⁹. New capsids with different parentage and reducedsusceptibility to neutralization are needed to enable future liver genetherapy trials with maximum patient enrollment. Few antigenic epitopeshave been characterized for parental serotypes or shuffled variants,thus precluding rational design attempts that still maximize capsidlibrary diversity. Screening future capsid variants in preclinicalvalidations against pools of patient antibodies represents an unbiasedapproach to improve the ultimate utility of variants moving into theclinic.

An exciting possibility with the new capsid variants that transducehuman liver at such high levels would be to decrease patient doses whilestill enabling desired expression levels. This could bypass severalhurdles to rAAV being an effective vector for future liver gene therapytrials: a) reduced probability for neutralization as fewer circulatingcapsids would be present; b) reduced production costs to offset thestaggering treatment costs that can reach $1 million dollars perpatient^(36,37); and c) reduced probability for capsid-specific T-cellresponses against transduced hepatocytes³⁸. Similarly, high-levelfunctional transduction will be key for the field as those in the fieldcollectively transition from treating simple non-cell autonomous liverdiseases like Crigler-Najjar and the hemophilias, to cell-autonomousdiseases like ornithine transcarbamylase deficiency and other urea cycledisorders which will require at least 20-50% of human hepatocytes to befunctionally transduced for clinical efficacy.

Methods

Shuffled AAV Capsid Plasmid Library Generation.

The shuffled AAV capsid library was generated as described previously⁴with modifications described below. The AAV capsid genes from serotypes1, 2, 3b, 4, 5, 6, 8, 9_hu14, avian and bovine were PCR-amplified withhigh-fidelity polymerase and cloned using a Zero Blunt TOPO PCR CloningKit (Invitrogen Cat#K2800) followed by Sanger sequencing of individualclones. Capsid genes were excised, mixed at 1:1 ratios and digestedusing DNaseI at various intervals from 1-30 min. These pooled reactionswere separated on a 1% (w/v) agarose gel, and fragments <1,000-bp wereexcised and used in a primer-less PCR reassembly step, followed by asecond round of PCR using primers binding outside the capsid gene:

(SEQ ID NO: 14) Fwd: 5′-GTCTGAGTGACTAGCATTCG-3′ (SEQ ID NO: 15)Rev: 5′-GCTTACTGAAGCTCACTGAG-3′

Full-length shuffled capsid genes were cloned into a modified pAAV2 hostplasmid (ITR-Rep2-Cap cloning site-AAV2 3′UTR sequence-ITR) withSwaI/NsiI restriction sites flanking the CAP insertion site and amodified portion of the AAV2 VP1 3′ UTR (see FIG. 18). Ligations weretransformed into numerous independent electro-competent cell aliquotsand diluted 1:40 in LB culture with low ampicillin (50-g/mL) for minimalexpansion. An aliquot was plated, and 100 clones were picked and Sangersequenced to validate library diversity. The pool of library plasmidswas purified using an EndoFree Plasmid Mega Kit (Qiagen Cat#123811) andused to produce libraries of replication-competent AAV virions.

AAV Library Production, Vector Production and Titration.

AAV library productions were produced using a Ca₃(PO₄)₂ transfectionprotocol (wtAAV library plasmid pool and pAdS helper) in HEK293T cells(ATCC Cat#CRL-3216) followed by double cesium chloride density gradientpurification, dialysis as previously described³⁹, and resuspended indPBS with 5% sorbitol (w/v) and 0.001% Pluronic F-68 (v/v). AAVlibraries were titered for Rep by TaqMan qPCR with the followingprimer/probe set:

(SEQ ID NO: 16) Fwd: 5′-TTCGATCAACTACGCAGACAG-3′ (SEQ ID NO: 17) Rev:5′-GTCCGTGAGTGAAGCAGATATT-3′ (SEQ ID NO: 18) Probe:5′/FAM/TCTGATGCTGTTTCCCTGCAGACA/BHQ-1/-3′

Recombinant AAV vector productions were similarly produced as above butas triple transfections (PEI or Ca₃(PO₄)₂) with pAdS helper, AAVtransfer vector (ssAAV-CAG-GFP-WPRE-SV40 pA from Addgene #37825;ssAAV-EF1α-FLuc-WPRE-HgHpA cloned in-house; ssAAV-LSP1-GFP-WPRE-BgHpAfrom Ian Alexander⁴⁰), and pseudotyping plasmids for each capsid ofinterest. AAV-GFP vectors were titered on GFP by TaqMan qPCR with thefollowing primer/probe set:

(SEQ ID NO: 19) Fwd: 5′-GACGTAAACGGCCACAAGTT-3′ (SEQ ID NO: 20) Rev:5′-GAACTTCAGGGTCAGCTTGC-3′ (SEQ ID NO: 21) Probe:5′/FAM/CGAGGGCGATGCCACCTACG/BHQ-1/-3′

AAV-FLuc vectors were titered by TaqMan qPCR with the followingprimer/probe set:

(SEQ ID NO: 22) Fwd: 5′-CACATATCGAGGTGGACATTAC-3′ (SEQ ID NO: 23) Rev:5′-TGGTTTGTATTCAGCCCATAG-3′ (SEQ ID NO: 24) Probe:5′/FAM/ACTTCGAGATGAGCGTTCGGCTG/BHQ-1/-3′

Mice.

Fah/Rag2/Il2rgc (FRG) deficient mice²⁵ on a C57BL/6J background and FRGmice on a NOD-strain background (FRGN) were housed and maintained inspecific-pathogen-free barrier facilities at either Oregon Health &Science University (U.S.), Stanford University (U.S.), or the Children'sMedical Research Institute (Australia). FRG/N mice were maintained onirradiated high-fat low-protein mouse chow (U.S.: Lab DietCat#Picolab-5LJ5; Australia: Specialty Feeds Cat#5415-024) ad libitum todecrease flux through the tyrosine pathway. Beginning on the day oftransplantation, FRG/N mice in the U.S. were maintained on 1-week ofacidified water to prevent bacterial growth, while mice in Australiareceived acidified water supplemented with 25-mg/mL Baytril antibiotic.The following week, mice in the U.S. were switched to 1-week of 8-mg/LSMX-TMP antibiotic water (supplemented with 0.7-mol/L dextrose forpalatability), while mice in Australia were switched to 1-mg/L NTBCwater. Thereafter, at each location, FRG/N mice were cycled on and off1-mg/L NTBC water as described^(25,41). Adult Balb/cJ mice werepurchased from The Jackson Laboratories (Cat#00651) for imaging studies.The Institutional Animal Care & Use Committees of Stanford University,Oregon Health & Science University and the Children's Medical ResearchInstitute approved all mouse procedures.

Hepatocyte Transplantation.

Donor human hepatocytes for transduction studies were acquired fromeither Celsis (Cat#F00995, Lot#LTS) from a 17-year-old Caucasian femalefor U.S. studies, or from Lonza (Cat#CC-25915, Lot#9F3097) from aCaucasian male for Australian studies. Weanling FRG/N mice werepre-conditioned with administration of recombinant human adenovirusexpressing urokinase (1.25E9 PFU by tail vein in Australia and 5E10 PFUretroorbitally in U.S.) 24-hr prior to transplant to promote human cellengraftment. 5E5-1E6 human hepatocytes were injected intrasplenicallyinto anesthetized recipient FRG/N mice and cycled on/off NTBC to promotehuman hepatocyte engraftment and expansion²⁵. Broad-spectrum antibiotic(Ceftiofur 4-mg/kg in U.S.; Baytril 5.6-mg/kg in Australia) was given byintraperitoneal injection immediately prior to surgery and for two daysfollowing surgery. Several weeks post-transplant, circulating humanalbumin levels were used to assess percent human engraftment fromseveral microliters of peripheral mouse blood.

Human Albumin ELISA.

To assess percent human engraftment in chimeric mice, severalmicroliters of peripheral blood were used to measure human albumin usingthe Bethyl Quantitative Human Albumin ELISA kit (Cat#E88-129) permanufacturer's protocol. This same kit was used to detect secreted humanalbumin levels in the media supernatant during human hepatic organoiddifferentiation as a marker of successful differentiation.

Replication-Competent AAV Library Selection in Humanized FRG Mice.

Female FRG mice with confirmed humanization were transduced with 5E10vg/mouse of AAV library by intravenous tail vein administration. 5E9 PFUof wild-type replication competent human Adenovirus-5 (hAd5) (ATCCCat#VR-5) in 20-μl volume was administered by intravenous retroorbitalinjection 24-hr later. Transduced humanized livers were harvested 48-hrafter hAd5 administration. Livers were minced, subjected to threefreeze-thaw cycles and further homogenization to ensure complete lysisof remaining hepatocytes. Liver lysates were then subjected to 65° C.for 30-min to heat inactivate the hAd5, and spun at 14,000 RPM at 4° C.to separate viral-containing supernatants from cellular debris. Viralsupernatants were dot-blot titered after each round to ensure continualadministration of 5E10 vg/mouse at each subsequent round of in vivoselection for a total of 5 rounds.

Sequential Sub-Screen on Evolved Human-Liver Library Against PooledHuman Immunoglobulins.

PureProteome protein-G magnetic beads (Millipore Cat#LSKMAGG02) werepre-loaded with pooled human immunoglobulins (Baxter Gammagard IVIGLiquid™, Cat #LE1500190, Lot #LE12J338AB) for 60-min at 4° C. per beadmanufacturer instructions for direct immunoprecipitation protocols. TheAAV library from round 5 of the in vivo screen was applied to theIgG-loaded beads for 12-hr rotating at 4° C. Bound and unbound fractionswere natively eluted per manufacturer instructions and run over a newset of IgG-loaded beads to enrich for true IgG-bound and unbound AAVpopulations. Viral gDNA was extracted from each fraction using theMinElute Virus Spin Kit (Qiagen Cat#57704), followed by PCRamplification using:

(SEQ ID NO: 25) Fwd: 5′-TGGATGACTGCATCTTTGAA-3′ (SEQ ID NO: 26)Rev: 5′-TGCTTACCCGGGTTACGAGTCAGGTATCTG-3′

AAV capsid ORFs from round 2 of the subscreen for IgG evasion werecloned using a Zero Blunt TOPO Kit and 100 clones were sent for fullSanger sequencing to assess diversity with primers:

(SEQ ID NO: 27) Fwd-1: 5′-TGGATGACTGCATCTTTGAA-3′ (SEQ ID NO: 28)Fwd-2: 5′-ATTGGCATTGCGATTCC-3′

Vectorization and Sequence Contribution Analysis of Evolved AAV Capsids.

Contigs were assembled using Geneious R7 v7.1.9 software and clonesselected for vectorization were amplified using:

(SEQ ID NO: 30) Fwd: 5′-AAATCAGGTATGGCTGCCGATG-3′ (SEQ ID NO: 31)Rev: 5′-GCTTCCCGGGATGGAAACTAGATAAGAAAG-3′

PCR amplicons were cloned in-frame, downstream of Rep, into predigestedrecipient pCap packaging plasmid containing AAV2 Rep without ITRs usingSwaI and XmaI restriction sites. AAV capsid genes were sequence verifiedand resultant contigs were analyzed using a custom Perl pipeline thatassesses multiple sequence alignments using Clustal Omega (EMBL-EBI) togenerate the overall serotype composition of the shuffled AAVs bycomparison of DNA and amino acid sequences with the parental AAVserotypes based on maximum likelihood. Xover 3.0 DNA/protein shufflingpattern analysis software was used to generate parental fragmentcrossover maps of shuffled variants⁴². Each parental serotype was colorcoded as follows: AAV1: red; AAV2: forest green; AAV3b: marine blue;AAV4: magenta; AAV5: tv blue; AAV6: green cyan; AAV8: orange; AAV9: palegreen; avian: purple; bovine: deep salmon).

PacBio Library Preparation and Full-Length Single-Molecule CapsidSequencing.

Pacific Biosciences (PacBio) SMRT bell libraries were prepared followingthe “Procedure and Checklist-2 kb Template Preparation and Sequencing”protocol from PacBio using the SMRTbell Template Prep Kit v1.0 (PacBioCat#100-259-100). PacBio ‘Binding and Annealing’ calculator was used todetermine appropriate concentrations for annealing and binding ofSMRTbell libraries. SMRTbell libraries were annealed and bound to P6 DNApolymerase for sequencing using the DNA/Polymerase Binding Kit P6 v2.0(PacBio Cat#100-372-700). Bound SMRTbell libraries were loaded onto SMRTcells using standard MagBead protocols and the MagBead Buffer Kit v2.0(PacBio Cat#100-642-800). The standard MagBead sequencing protocol wasfollowed with the DNA Sequencing Kit 4.0 v2 (PacBio Cat#100-612-400,also known as P6/C4 chemistry). Sequencing data was collected for 6-hourmovie times with ‘Stage Start’ not enabled. Circular consensus sequence(CCS) reads were generated using the PacBio SMRT portal and theRS_ReadsOfinsert.1 protocol, with filtering set at Minimum Full Pass=3and Minimum Predicted Accuracy=95%.

Bioinformatic Assessment of PacBio Sequence Reads.

CCS reads with sequence lengths from 2,300-2,350 nucleotides wereincluded in downstream bioinformatics analyses. Indels in CCS reads werecorrected using an in-house algorithm that first assesses parentalfragment identity to determine correct parental nucleotide sequences tocompare for determining indels for correction. Single nucleotidepolymorphisms that did not result in indels, were maintained. Correctedsequences in FASTA format were then aligned with MUSCLE⁴³. Phylogeneticanalyses were conducted using the maximum-likelihood method in RAxML⁴⁴.Percent parental conservation was determined using an in-house algorithmthat identifies the percentage of each parent on each aligned positionin the shuffled library. The maximum square size indicates that 100% ofvariants share that amino acid from that parent at that position. Allother square sizes are proportional to the percent of variants from0-100% that have that amino acid at that position from that parent.

Transduction Mouse Experiments.

All mice received normodynamic intravenous lateral tail vein injectionsof 2E11 vg/mouse of ssAAV-CAG-GFP or ssAAV-LSP1-GFP pseudotyped withvarious capsid serotypes. Treated mice were monitored for 14-days andlivers were harvested under inhalation isoflurane anesthesia. Livertissue was cut into several 2×5-mm pieces from several lobes and fixedin 10× volume of 4% PFA for 5-hr at 25° C. protected from light. Fixedtissue was washed 1× in PBS and put through a sucrose cryoprotection andrehydration series (10% w/v sucrose for 2-hr at 25° C., 20% w/v sucroseovernight at 4° C., 30% w/v sucrose for 4-hr at 25° C.). Liver pieceswere rinsed in PBS, blotted dry and mounted in cryomolds (Tissue-TekCat#4557) with OCT (Tissue-Tek Cat#4583) and frozen in a liquidnitrogen-cooled isopentane bath. Cryomolds were placed at −80° C. untilsectioning.

Liver Immunohistochemistry.

Fluorescent staining of liver sections for human FAH was performed perestablished protocols⁴⁵ with minor modifications. Modificationsincluded: fixing slides in ice-cold methanol for 10 minutes rather thanacetone at room temperature; blocking with 10% rather than 5% donkeyserum (Santa Cruz, Cat#sc-2044) in dPBS for 30-min at RT in a humidifiedchamber; primary antibody solution was 100-μl of monoclonal rabbitanti-human FAH IgG antibody (Sigma, Cat#HPA-04137) at 1:100 (Australia)or 1:500 (U.S.) in 10% donkey serum incubated overnight at 4° C. (U.S.)or for 2-hrs at RT (Australia); secondary antibody solution was 100-μlof donkey anti-rabbit AlexaFluor 647 IgG antibody (Invitrogen,Cat#A31573) at 1:500 along with Hoechst 33342 (Molecular Probes,Cat#H-3570) at 1:1,000 in PBST for 1-hr at RT in dark conditions (U.S.),or 100-μl of donkey anti-rabbit AlexaFluor 594 IgG antibody (Invitrogen,Cat#A21207) at 1:500 in PBS for 1-hr at RT in dark conditions followedby DAPI at 80-ng/mL in PBS; slides were mounted with 3 drops of ProLongGold Antifade (Invitrogen, Cat#P36934) (U.S.) or ProLong DiamondAntifade (Invitrogen, Cat#P36961) (Australia). Antibody validitycontrols included secondary-only staining, and demonstration on positivecontrol frozen human liver tissue sections (Zyagen, Cat#HF-314) andnegative control frozen untreated mouse liver sections. Confocal imagingin the U.S. was performed on a Leica TCS SP8-X WLL inverted confocalmicroscope with a 20× oil immersion objective and imaged with Leica AFsoftware v3.3.0.10134, while confocal imaging in Australia was performedon an inverted Zeiss Axio with 20× objective and imaged with Zen Prosoftware. Z-stacks were compressed using ImageJ v2.0.0 and overlaid inAdobe Photoshop CS6 v13.0. Signal co-localization of AAV-GFP signal withmouse or human hepatocytes was done using Volocity v6.3 software andre-validated with counts by eye on a subset of sections.

Hepatic Fluorescent In Situ Hybridization (FISH).

Sequential RNA and DNA FISH on OCT-embedded frozen liver sections fromtreated humanized mice was performed as described⁴⁶. To localize RNAFISH signals, slides were analyzed by acquiring multiple 3D images,recording coordinates of all imaged fields, and combining planes fromeach field using an EDF (extended depth of focus) function into a seriesof single-focused images for each imaged field. Subsequent DNA FISH wascompleted as described⁴⁶ and the previously imaged fields were imagedagain, in the same manner. Comparing the image sets allows one todetermine the relative position of RNA and DNA signals. The addition ofGFP immunostaining showed the relationship between transcription andtranslation of AAV transfer vector DNA. Images were taken on a NikonEclipse E800 wide-field microscope (60× Plan Apochromat objective with1.4-NA) with a Photometrics Coolsnap ES camera and Nikon NIS Elementssoftware v4.2.

Functional Validation of Human Hepatic Organoid Cultures.

Human liver non-parenchymal cells from a 23-year-old male were culturedas described^(47,48) with minor modifications (gastrin was omitted andALKS inhibitor SB431542 was added). To functionally demonstrate hepaticorigin of organoids, media from organoid cultures was tested for thepresence of human albumin by ELISA (human albumin=58.3-ng/mL).

Human Hepatic Organoid Transduction with AAV.

Initiated hepatic organoid cultures were passaged at a ratio of 1:4 intostandard organoid conditions (embedding in >95% Matrigel followed byaddition of liquid media) in 24-well suspension plates after two weeksof growth. After the fifth organoid passage, ssAAV-CAG-GFP preparationsof each serotype were added at MOI 500K to each well. Media was changedafter a 3-day incubation and daily thereafter, and the emergence of GFPexpression was monitored daily by fluorescence microscopy andbrightfield imaging. After 14 days of imaging on an EVOS F1 ImagingSystem (ThermoFisher Cat#AMF4300), single hepatic cells were dissociatedfrom the organoid with TrypLE Express (ThermoFisher, Cat#1797945) and100K dissociated cells were analyzed for GFP positivity by flowcytometry (BD Canto II).

Indirect Seroreactivity ELISA Assay for Anti-AAV Antibodies in HumanSerum.

Off-clot serum collected from peripheral blood of 50 healthy U.S. adults(see Table 4) was used as the primary antibody in an indirect ELISA.Pooled human IgG (Baxter, Cat#LE1500190, Lot#LE12P180AB) from thousandsof donors was used to prepare a standard curve (sixteen 2-fold dilutionsof 100-mg/ml stock IVIG in blocking buffer). Shuffled and parental AAVcapsids served as antigens (5E8 vector genomes/well). Human IgGstandards were assessed in replicates of six and all AAV samples wereassessed in triplicate. IgG standards and AAV samples were fixed towells of a 96-well immunoplate with 50-μl coating solution (13 mMNa₂CO₃, 35 mM NaHCO₃ in water, pH 9.6), plates were sealed and incubatedovernight at 4° C. Plates were washed 2× with PBST containing 0.05%Tween-20 and blocked with blocking buffer (PBS, 6% BSA, 0.05% Tween-20)for 1-hr at 25° C. Plates were washed 2× with PBST. Each of the 50 humansera samples was diluted in blocking buffer (1:100-1:2,000), and 50-μlwas added to experimental wells. Plates were incubated for 2-hr at 37°C. and then washed 2× in PBST. Polyclonal sheep anti-human IgG-HRPsecondary antibody (GE Bioscience Cat#NA933V) was diluted 1:500 in washbuffer and 100-μl added to each well to detect bound antibodies in thehuman sera. Plates were incubated for 2-hr at 37° C. and washed 2× inPBST. OPD substrate (o-phenylenediaminedihydrochloride, Sigma Cat#P4664)was added at 100-μl/well in a 0.1M sodium citrate buffer and plates wereincubated at 25° C. for exactly 10-min. The reaction was stopped with50-4/well of 3M H₂SO₄ and the absorbance determined at 490-nm on amicroplate reader (Bio-Rad). A set of blank wells was used to subtractbackground for non-specific binding of antibodies to the immunoplate.Standards were plotted using Four Parameter Logistic curve fitting todetermine sample concentrations that fall within the linear range of thedilution series and detection limits using Prism v6.0 software. The sameassay was performed on a cohort of 21 adult males with hemophilia B (seeTable 9).

Indirect Seroreactivity ELISA Assay for Anti-AAV Antibodies in NormalRhesus Macaque Serum.

The seroreactivity ELISA was performed as previously described⁴⁹ withplates coated at 1E9 vp/well. Off-clot serum was collected fromperipheral blood from 6 rhesus macaques (see Table 6).

Luminescence-Based AAV Neutralization Assay with Individual Human SerumSamples.

The neutralization assay was performed as previous described⁵⁰. Off-clotserum was collected from peripheral blood of 21 healthy E.U. individuals(see Table 8). ssAAV-CMV-FLuc vector was used as the transfer vector atan MOI of 200.

False-Colored Structural Capsid Mapping.

Chimeric capsids were false-color mapped onto the AAV2 capsid structure1LP3⁵¹ using Pymol v1.7.6.0. Mapped colors correspond to parentalserotype colors used in the parental fragment crossover maps. Exteriorcapsid views have all chains represented, while cross-section views havechains surrounding a cylinder at the 5-fold symmetry axis removedexposing the capsid interior lumen.

Statistics.

Statistical analyses were conducted with Prism v6 and Excel v14.5.8software. Experimental values for each panel in FIG. 15 were log+1transformed before being assessed via two-way ANOVA using Tukey'smultiple comparisons test. P values <0.05 were considered statisticallysignificant. Additional experimental differences were evaluated using aStudent's unpaired two-tailed t-test assuming equal variance.

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The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

What is claimed is:
 1. An adeno-associated virus (AAV) vector comprisinga nucleic acid sequence encoding a variant AAV capsid polypeptide,wherein said variant AAV capsid polypeptide exhibits increasedtransduction or tropism in human liver tissue or hepatocyte cells ascompared to a non-variant parent capsid polypeptide, wherein saidvariant AAV capsid polypeptide sequence comprises a sequence selectedfrom the group consisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ IDNO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).
 2. The AAVvector of claim 1, wherein said variant AAV capsid polypeptide sequenceis AAV-NP84 (SEQ ID NO:2).
 3. The AAV vector of claim 1, wherein saidvariant AAV capsid polypeptide sequence is AAV-NP59 (SEQ ID NO:4). 4.The AAV vector of claim 1, wherein said variant AAV capsid polypeptidesequence is AAV-NP40 (SEQ ID NO:6).
 5. The AAV vector of claim 1,wherein said variant AAV capsid polypeptide sequence is AAV-NP30 (SEQ IDNO:8).
 6. The AAV vector of claim 1, wherein said nucleic acid sequenceencoding said variant AAV capsid polypeptide comprises a sequenceselected from the group consisting of AAV-NP84 (SEQ ID NO:1), AAV-NP59(SEQ ID NO:3), AAV-NP40 (SEQ ID NO:5), and AAV-NP30 (SEQ ID NO:7).
 7. Amethod of using the AAV vector of claim 1, in a therapeutic treatmentregimen or vaccine.
 8. A method of using the AAV vector of claim 1 toreduce the amount of total AAV vector administered to a subject, saidmethod comprising administering less total AAV vector amount to saidsubject when said AAV vector is transduced by a variant AAV capsidpolypeptide as compared to the amount of AAV vector administered to saidsubject when said AAV vector is transduced by a non-variant parentcapsid polypeptide in order to obtain a similar therapeutic effect. 9.An adeno-associated virus (AAV) vector comprising a nucleic acidsequence encoding a variant AAV capsid polypeptide, wherein said variantAAV capsid polypeptide exhibits an enhanced neutralization profile ascompared to a non-variant parent capsid polypeptide, wherein saidvariant AAV capsid polypeptide exhibits increased transduction of humanliver organoids in 3-dimensional cultures in vitro as compared to anon-variant parent capsid polypeptide, wherein said variant AAV capsidpolypeptide sequence comprises a sequence selected from the groupconsisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ ID NO:4), AAV-NP40(SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).
 10. The AAV vector of claim1, wherein said variant AAV capsid polypeptide exhibits an enhancedneutralization profile against pooled human immunoglobulins as comparedto a non-variant parent capsid polypeptide.
 11. The AAV vector of claim1, wherein said variant AAV capsid polypeptide exhibits increasedtransduction or tropism in human liver tissue or hepatocyte cells. 12.The AAV vector of claim 1, wherein said variant AAV capsid polypeptideexhibits increased transduction as compared to a non-variant parentcapsid polypeptide.
 13. The AAV vector of claim 1, wherein said variantAAV capsid polypeptide exhibits increased tropism as compared to anon-variant parent capsid polypeptide.
 14. The AAV vector of claim 1,wherein said variant AAV capsid polypeptide further exhibits increasedtransduction or tropism in one or more non-liver human tissues ornon-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.
 15. The AAV vector of claim 1, wherein said variant AAVcapsid polypeptide exhibits increased transduction of human liver tissueor hepatocyte cells in vivo as compared to a non-variant parent capsidpolypeptide.
 16. The AAV vector of claim 1, wherein said variant AAVcapsid polypeptide exhibits increased transduction of human liver tissueor hepatocyte cells in vitro as compared to a non-variant parent capsidpolypeptide.
 17. An adeno-associated virus (AAV) vector comprising anucleic acid sequence encoding a variant AAV capsid polypeptide, whereinsaid variant AAV capsid polypeptide exhibits an enhanced neutralizationprofile as compared to a non-variant parent capsid polypeptide, whereinsaid vector further comprises a nucleic acid sequence comprising atherapeutic expression cassette, wherein said expression cassette is aCRISPR/CAS expression system, wherein said variant AAV capsidpolypeptide sequence comprises a sequence selected from the groupconsisting of AAV-NP84 (SEQ ID NO:2), AAV-NP59 (SEQ ID NO:4), AAV-NP40(SEQ ID NO:6), and AAV-NP30 (SEQ ID NO:8).
 18. The AAV vector of claim17, wherein said therapeutic expression cassette encodes a therapeuticprotein or antibody.
 19. An adeno-associated virus (AAV) vectorcomprising a nucleic acid sequence encoding a variant AAV capsidpolypeptide, wherein said variant AAV capsid polypeptide exhibits anenhanced neutralization profile as compared to a non-variant parentcapsid polypeptide, wherein said variant AAV capsid polypeptide sequencecomprises a sequence selected from the group consisting of AAV-NP84 (SEQID NO:2), AAV-NP59 (SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30(SEQ ID NO:8).
 20. The AAV vector of claim 19, wherein said variant AAVcapsid polypeptide sequence is AAV-NP84 (SEQ ID NO:2).
 21. The AAVvector of claim 19, wherein said variant AAV capsid polypeptide sequenceis AAV-NP59 (SEQ ID NO:4).
 22. The AAV vector of claim 19, wherein saidvariant AAV capsid polypeptide sequence is AAV-NP40 (SEQ ID NO:6). 23.The AAV vector of claim 19, wherein said variant AAV capsid polypeptidesequence is AAV-NP30 (SEQ ID NO:8).
 24. An adeno-associated virus (AAV)vector comprising a nucleic acid sequence encoding a variant AAV capsidpolypeptide, wherein said nucleic acid sequence encoding said variantAAV capsid polypeptide comprises a sequence selected from the groupconsisting of AAV-NP84 (SEQ ID NO:1), AAV-NP59 (SEQ ID NO:3), AAV-NP40(SEQ ID NO:5), and AAV-NP30 (SEQ ID NO:7).
 25. A method of using the AAVvector of claim 24 in a therapeutic treatment regimen or vaccine.
 26. Amethod of using the AAV vector of claim 24 to reduce the amount of totalAAV vector administered to a subject, said method comprisingadministering less total AAV vector amount to said subject when said AAVvector is transduced by a variant AAV capsid polypeptide as compared tothe amount of AAV vector administered to said subject when said AAVvector is transduced by a non-variant parent capsid polypeptide in orderto obtain a similar therapeutic effect.
 27. The AAV vector of claim 24,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP84 (SEQ ID NO:1).
 28. The AAV vector of claim 24,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP59 (SEQ ID NO:3).
 29. The AAV vector of claim 24,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP40 (SEQ ID NO:5).
 30. The AAV vector of claim 24,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP30 (SEQ ID NO:7).
 31. The AAV vector of claim 6,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP84 (SEQ ID NO:1).
 32. The AAV vector of claim 6,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP59 (SEQ ID NO:3).
 33. The AAV vector of claim 6,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP40 (SEQ ID NO:5).
 34. The AAV vector of claim 6,wherein said nucleic acid sequence encoding said variant AAV capsidpolypeptide is AAV-NP30 (SEQ ID NO:7).
 35. The AAV vector of claim 24,wherein said variant AAV capsid polypeptide exhibits an enhancedneutralization profile against pooled human immunoglobulins as comparedto a non-variant parent capsid polypeptide.
 36. The AAV vector of claim24, wherein said variant AAV capsid polypeptide exhibits increasedtransduction or tropism in human liver tissue or hepatocyte cells. 37.The AAV vector of claim 24, wherein said variant AAV capsid polypeptideexhibits increased transduction as compared to a non-variant parentcapsid polypeptide.
 38. The AAV vector of claim 24, wherein said variantAAV capsid polypeptide exhibits increased tropism as compared to anon-variant parent capsid polypeptide.
 39. The AAV vector of claim 24,wherein said variant AAV capsid polypeptide further exhibits increasedtransduction or tropism in one or more non-liver human tissues ornon-hepatocyte human cells as compared to a non-variant parent capsidpolypeptide.
 40. The AAV vector of claim 24, wherein said variant AAVcapsid polypeptide exhibits increased transduction of human liver tissueor hepatocyte cells in vivo as compared to a non-variant parent capsidpolypeptide.
 41. The AAV vector of claim 24, wherein said variant AAVcapsid polypeptide exhibits increased transduction of human liver tissueor hepatocyte cells in vitro as compared to a non-variant parent capsidpolypeptide.
 42. An adeno-associated virus (AAV) vector comprising anucleic acid sequence encoding a variant AAV capsid polypeptide, whereinsaid variant AAV capsid polypeptide exhibits increased transduction ortropism in human liver tissue or hepatocyte cells as compared to anon-variant parent capsid polypeptide, wherein said variant AAV capsidpolypeptide exhibits increased transduction of human liver organoids in3-dimensional cultures in vitro as compared to a non-variant parentcapsid polypeptide, wherein said variant AAV capsid polypeptide sequencecomprises a sequence selected from the group consisting of AAV-NP84 (SEQID NO:2), AAV-NP59 (SEQ ID NO:4), AAV-NP40 (SEQ ID NO:6), and AAV-NP30(SEQ ID NO:8.
 43. The AAV vector of claim 42, wherein said variant AAVcapsid polypeptide exhibits an enhanced neutralization profile.
 44. TheAAV vector of claim 42, wherein said variant AAV capsid polypeptideexhibits an enhanced neutralization profile against pooled humanimmunoglobulins as compared to a non-variant parent capsid polypeptide.45. The AAV vector of claim 42, wherein said variant AAV capsidpolypeptide exhibits increased transduction or tropism in human livertissue or hepatocyte cells.
 46. The AAV vector of claim 42, wherein saidvariant AAV capsid polypeptide exhibits increased transduction ascompared to a non-variant parent capsid polypeptide.
 47. The AAV vectorof claim 42, wherein said variant AAV capsid polypeptide exhibitsincreased tropism as compared to a non-variant parent capsidpolypeptide.
 48. The AAV vector of claim 42, wherein said variant AAVcapsid polypeptide further exhibits increased transduction or tropism inone or more non-liver human tissues or non-hepatocyte human cells ascompared to a non-variant parent capsid polypeptide.
 49. The AAV vectorof claim 42, wherein said variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells in vivoas compared to a non-variant parent capsid polypeptide.
 50. The AAVvector of claim 42, wherein said variant AAV capsid polypeptide exhibitsincreased transduction of human liver tissue or hepatocyte cells invitro as compared to a non-variant parent capsid polypeptide.